Chemiluminescent probes for diagnostics and in vivo imaging

ABSTRACT

The present invention provides dioxetane-based chemiluminescence probes, more specifically fluorophore-tethered dioxetane-based chemiluminescence probes and compositions thereof. The chemiluminescence probes disclosed are useful for both diagnostics and in vivo imaging.

TECHNICAL FIELD

The present invention provides various dioxetane-based chemiluminescenceprobes and compositions thereof.

Abbreviations: AcOH, acetic acid; ACN, acetonitrile; BBBPY,4,4′-di-tert-butyl-2,2′-dipyridyl; CIEEL, chemically initiated electronexchange luminescence; DCM, dichloromethane; DEAD, diethylazodicarboxylate; DMAP, 4-dimethylaminopyridine; DMF,N,N′-dimethylformamide; DMSO, dimethyl sulfoxide; ET, energy transfer;EtOAc, ethylacetate; Hex, hexane; LDA, lithium diisopropylamide; MeOH,methanol; NHS, N-hydroxysuccinimide; NIR, near-infrared; NIS,N-iodosuccinimide; PBS, Phosphate buffered saline; QCy, quinone-cyanine;RP-HPLC, reverse-phase high pressure liquid chromatography; RLU,relative light unit; RT, room temperature; TBAF, tetra-n-butylammoniumfluoride; TBDMS, tert-butyldimethylsilyl; TBDPS,tert-butyldiphenylsilyl; TBS, tert-butyldimethylsilyl; TBSCl,tert-butyldimethylsilyl chloride; TEMP, 2,2,6,6-tetramethylpiperidine;TFA, trifluoroacetic acid; THF, tetrahydrofuran; TLC, thin layerchromatography; TMS-Cl, trimethylsilyl chloride.

BACKGROUND ART

Chemiluminescence assays are widely utilized in various chemical andbiological applications due to their sensitivity and highsignal-to-noise ratio (Roda and Guardigli, 2012; Roda et al., 2005).Unlike fluorescence-based assays, in chemiluminescence no lightexcitation is required. Therefore, background signal arising fromautofluorescence does not exist when chemiluminescence is used. Suchcircumstance makes chemiluminescence especially useful for tissue andwhole-body imaging (Gross et al., 2009; Zhang et al., 2013; Van deBittner et al., 2013; Porterfield et al., 2015).

Most of the chemiluminescent compounds, currently in use, are activatedby oxidation; i.e., a stable precursor is oxidized usually by hydrogenperoxide, to form an oxidized high-energy intermediate, which thendecomposes to generate an excited species. The latter decays to itsground state by either light emission or by energy transfer. Commonprobes that act on such chemiluminescence mechanism are usually based onluminol (Merényi et al., 1990) and oxalate esters (Silva et al., 2002).Utilizing this oxidation-activated chemiluminescence mode-of-action,several systems were developed for the in vivo imaging of reactiveoxygen species (ROS) (Lee et al., 2007; Kielland et al., 2009; Lim etal., 2010; Cho et al., 2012; Lee et al., 2012; Shuhendler et al., 2014;Lee et al., 2016; Li et al., 2016).

Innately, chemiluminescence that is exclusively activated by oxidationis limited for the detection and imaging of ROS. However, in 1987 PaulSchaap developed a new class of chemiluminescent probes, which can beactivated by an enzyme or an analyte of choice (Schaap et al., 1987a-c).As depicted in Scheme 1, Schaap's adamantylidene-dioxetane basedchemiluminescence probe (structure I) is equipped with ananalyte-responsive protecting group used to mask the phenol moiety ofthe probe. Removal of the protecting group by the analyte of interestgenerates an unstable phenolate-dioxetane species II, which decomposesthrough a chemiexcitation process to produce the excited intermediatebenzoate ester III and adamantanone. The excited intermediate decays toits ground-state (benzoate ester IV) through emission of a blue lightphoton.

In bioassays, under aqueous conditions, Schaap's dioxetanes suffer fromone major limitation; their chemiluminescence efficiency decreasessignificantly through non-radiative energy transfer processes(quenching) by interaction with water molecules (Matsumoto, 2004). Acommon way to amplify the chemiluminescence signal of Schaap'sdioxetanes is achieved through energy transfer from the resultingexcited species (benzoate ester III) to a nearby acceptor, which is ahighly emissive fluorophore under aqueous conditions (Park et al., 2014;Tseng and Kung, 2015). Therefore, a surfactant-dye adduct is usuallyadded in commercial chemiluminescent immunoassays. The surfactantreduces water-induced quenching by providing a hydrophobic environmentfor the excited chemiluminescent probe, which transfers its energy toexcite a nearby fluorogenic dye. Consequently, the low-efficiencyluminescence process is amplified up to 400-fold in aqueous medium(Schaap et al., 1989). However, since the surfactant mode-of-actionrelies on micelles formation, its functional concentration is relativelyhigh (above the critical micelle concentration) (Dominguez et al.,1997). As micellar structures are not maintained when animals aretreated systemically, the surfactant-dye adduct approach is notpractical for in vivo detection or imaging of biological activitygenerated by enzymes or chemical analytes (Torchilin, 2001).

To overcome the limitation of a two-component system (a dioxetane probeand a surfactant-fluorescent dye adduct), a single component comprisedof dioxetane conjugated with fluorophore is required. Two previousreports have described synthesis of dioxetane-fluorogenic dyeconjugates, in which the dioxetane effectively transferschemiluminescence energy to the tethered fluorophore to emit light at awavelength that can be varied by choice of fluorophore (WO 1990007511;Watanabe et al., 2012). In addition to signal amplification, tetheringof dioxetane with fluorophore also allows color modulation andred-shifting of the emitted light; a significant requirement forbioimaging applications (Matsumoto et al., 2008; Loening et al., 2010;Branchini et al., 2010; McCutcheon et al., 2012; Jathoul et al., 2014;Steinhardt et al., 2016).

SUMMARY OF INVENTION

Disclosed herein are two approaches for improving the chemiluminescenceof the Schaap's adamantylidene-dioxetane based chemiluminescence probe.

According to one approach wherein chemiluminescence emission isamplified through an indirect pathway (Study 1 herein), the Schaap'sadamantylidene-dioxetane based probe is conjugated to a fluorescent dyemeta to the analyte-responsive protecting group, via a linker. As shownin Scheme 2 (upper panel), the dioxetane-fluorophore conjugate thusobtained decomposes upon its activation to generate a benzoatederivative like intermediate V, and its chemiluminescent emission issignificantly amplified under physiological conditions through theenergy transfer mechanism from the excited benzoate to the fluorescentdye.

Study 1 shows a simple and practical synthetic route for preparation ofsuch fluorophore-tethered dioxetane chemiluminescent probes. Theeffectiveness of the synthesis is based on a late-stagefunctionalization of a dioxetane precursor by Hartwig-Miyaura C—Hborylation, followed by subsequent Suzuki coupling and oxidation todioxetane. The obtained intermediate is composed of a reactiveNHS-ester-dioxetane ready for conjugation with any fluorophore-aminederivative. The chemiluminescent emission of the fluorophore-tethereddioxetane probes was significantly amplified in comparison to a classicdioxetane probe through an energy transfer mechanism. The synthesizedprobes produced light of various colors that matched the emissionwavelength of the excited tethered fluorophore. Using the syntheticroute exemplified, two fluorophore-tethered dioxetane probes designedfor activation by β-galactosidase and conjugated with green(fluorescein) and NIR (QCy) fluorescent dyes were synthesized. Bothprobes were able to provide chemiluminescence in vivo images followingsubcutaneous injection after activation by β-galactosidase; however, achemiluminescence image following intraperitoneal injection was observedonly by the NIR probe. These are the first in vivo images produced bySchaap's dioxetane-based chemiluminescence probes with no need of anyadditive. The NIR probe was also able to image cells bychemiluminescence microscopy, based on endogenous activity ofβ-galactosidase.

The intermediates obtained following functionalization of a dioxetaneprecursor by Hartwig-Miyaura C—H borylation are referred to herein ascompounds of the formula I, and those obtained after further Suzukicoupling and oxidation are referred to herein as compounds of theformula IIa/IIb. The fluorophore-tethered dioxetane-basedchemiluminescence probes disclosed are referred to herein as compounds(or conjugates) of the formula IIIa/IIIb.

According to another approach wherein chemiluminescence emission isamplified through a direct mode of action (Study 2 herein), the Schapp'sadamantylidene-dioxetane probe is substituted at the ortho position ofthe phenolic ring with a π* acceptor group such as an acrylate andacrylonitrile electron-withdrawing group so as to increase the emissivenature of the benzoate species (Scheme 2, lower panel). To the best ofour knowledge, the influence of electron acceptor substituents on thearomatic moiety of dioxetane chemiluminescence probes was never studiedbefore for physiologically-relevant pHs.

Study 2 shows the preparation of such chemiluminescence probes with highefficiency yield under physiological conditions. The chemiluminescencequantum yield of the best probe was greater than three orders ofmagnitude in comparison to the standard commercially availableadamantylidene-dioxetane probe. Importantly, one of the probes preparedwas able to provide high quality chemiluminescence cell images based onendogenous activity of β-galactosidase, demonstrating for the first timecell-imaging achieved by non-luciferin small molecule based probe withdirect chemiluminescence mode of emission. The chemiluminescence probesshown in this Study are referred to herein as compounds of the formulaIVa/IVb.

In certain aspects, the present invention thus provides afluorophore-tethered dioxetane-based chemiluminescence probe of theformula IIIa/IIIb as defined herein, as well as intermediates for thepreparation thereof referred to herein as compounds of the formulas Iand IIa/IIb as defined herein; and a π* acceptor group-containingdioxetane based chemiluminescence probe of the formula IVa/IVb asdefined herein.

In a further aspect, the present invention provides a compositioncomprising a carrier, e.g., a pharmaceutically acceptable carrier, andeither a conjugate of the formula IIIa/IIIb or a compound of the formulaIVa/IVb. The composition of the invention may be used for diagnostics aswell as for in vivo imaging of reporter genes, enzymes, and chemicalanalytes.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows chemiluminescent emission spectrum of 1 μM Probe 1,λ_(max)=470 nm, recorded in PBS, pH 7.4, in the presence of 1.5 units/mLβ-galactosidase.

FIG. 2 shows decomposition of Probes 1, 2 and 3 under normal roomillumination conditions. Probes (300 μM) were incubated in PBS (100 mM),pH 7.4, at ambient temperature.

FIG. 3 illustrates a proposed pathway for visible-light-induceddecomposition of dioxetane-fluorophore conjugates.

FIG. 4 shows chemiluminescence emission spectra of 1 μM (panel A) 1:1mixture of Probe 1 and fluorescein derivative 2b, λ_(max)=470 nm, 535nm; (panel B) Probe 2, λ_(max)=535 nm; (panel C) 1:1 mixture of Probe 1and QCy derivative 3b, λ_(max)=470 nm; and (panel D) Probe 3,λ_(max)=714 nm. Spectra were recorded in PBS (100 mM), pH 7.4, in thepresence of 1.5 units/mL β-galactosidase (continuous lines). Dotted lineis the fluorescence emission spectrum (DCL—direct chemiluminescence).

FIG. 5 shows (panels A-C) chemiluminescent kinetic profiles of 1 μM (A)Probe 1, (B) Probe 2, and (C) Probe 3 in PBS (100 mM), pH 7.4, in thepresence of 1.5 units/mL 3-galactosidase and in the absence ofβ-galactosidase. Panels D-F show total photon counts emitted from (D)Probe 1, (E) Probe 2, and (F) Probe 3 in the presence ofβ-galactosidase.

FIG. 6 shows total light emitted from 10 μM Probe 2 in PBS (100 mM), pH7.4, over a period of 1 h, with different concentrations ofβ-galactosidase. The inset focuses on light emitted from Probe 2 uponincubation with the lowest concentrations of 3-galactosidase.

FIGS. 7A-7D show (7A) solution images obtained from 1 μM Probes 1, 2 and3 incubated in PBS, pH 7.4, in the presence and in the absence ofβ-galactosidase (gal); (7B) quantification of signal intensitiesobtained in solution in the presence of β-galactosidase; (7C) whole-bodyimages obtained 15 min following subcutaneous injection of Probes 2 and3 [50 μL, 1 μM in PBS (100 mM), pH 7.4, after 30 min pre-incubation withor without 1.5 units/mL β-galactosidase]; and (7D) quantification ofsignal intensities in whole-body images in the presence ofβ-galactosidase (quantitative data are based on repeated imagingexperiments with three mice).

FIG. 8 shows whole-body images obtained 15 min following intraperitonealinjection of Probes 2 and 3 [50 μL, 1 μM in PBS (100 mM), pH 7.4, aftera 30 min pre-incubation with or without 1.5 units/mL β-galactosidase].

FIG. 9 shows transmitted light image (panel a) and chemiluminescencemicroscopy of HEK293-LacZ stable cells (panel b); and transmitted lightimage (panel c) and chemiluminescence microscopy of HEK293-WT cells(panel d). Images were obtained following 20 min incubation with cellculture medium containing Probe 3 (5 μM).

FIG. 10 shows transmitted light image (panel a) and chemiluminescencemicroscopy of HEK293-LacZ stable cells, fixed with formaldehyde (4% for20 min) (panel b). Images were obtained following 20 min incubation withcell culture medium containing Probe 3 (5 μM).

FIG. 11 shows absorbance (O.D., solid line) and fluorescence (FLU,dashed line) spectra of benzoates 6a, 7a, 8a and 9a [50 μM] in PBS 7.4(5% DMSO), (excitation wavelength=290/400 nm).

FIGS. 12A-12B show chemiluminescence kinetic profiles of Probes 5, 6, 7,8 and 9 [1 μM] in PBS, pH 7.4 (10% DMSO) in the presence of 1.5 units/mLβ-galactosidase at room temp (12A; the inset shows the kinetic profileof Probe 5); and total emitted photons (12B).

FIGS. 13A-13B show chemiluminescence kinetic profiles of Probe 5 [1 μM]in the presence of 1.5 units/mL β-galactosidase, with and withoutEmerald-II™ enhancer (10%) in PBS 7.4 (10% DMSO) (left panel), and countof total emitted photon (right panel) (13A); and chemiluminescencekinetic profiles of Probe 5 [1 μM] with Emerald-II™ enhancer (10%) andProbe 9 [1 μM] in PBS 7.4 (10% DMSO) in the presence of 1.5 units/mLβ-galactosidase (13B) (left panel), and count of total emitted photon(right panel).

FIG. 14 shows water soluble chemiluminescence probes for detection ofhydrogen peroxide (Probe 10) and alkaline-phosphatase (AP) (Probe 11),which produce visible bright green luminescence under aqueousconditions. On the left—a comparison between light emission observed byProbe 10 (1 mM; left vial) to that observed by Luminol (1 mM; rightvial) upon incubation with hydrogen peroxide under aqueous conditions atpH 10. Probe 12 is a chemiluminescence for detection of GSH.

FIGS. 15A-15B show (15A) total light emitted from Probe 10 (100 μM),Probe 11 (10 μM) and Probe 12 (10 μM) in the presence of hydrogenperoxide (1 mM), alkaline phosphatase (AP) (1.5 EU/ml) or glutathione (1mM). Measurements were conducted in PBS (100 mM), pH 7.4, with 10% DMSOat RT; and (15B) total light emitted from Probe 10 (500 μM), Probe 11(500 μM) and Probe 12 (10 μM) in PBS (100 mM), pH 7.4 with 10% DMSO overa period of 1 h, with various concentration of the correspondingstimulus. A detection limit (blank control+3 SD) was determined for eachprobe.

FIG. 16 shows (a) transmitted light image and (b) chemiluminescencemicroscopy of HEK293-LacZ stable cells; and (c) transmitted light imageand (d) chemiluminescence microscopy of HEK293-WT cells. Images wereobtained following 20 min incubation with cell culture medium containingProbe 7 (5 μM). Images were taken by the LV200 Olympus-microscope using60× objective and 40 s exposure time.

DETAILED DESCRIPTION

In one aspect, the present invention provides a compound of the formulaI:

wherein

R¹ is selected from a linear or branched (C₁-C₁₈)alkyl, or(C₃-C₇)cycloalkyl;

R² and R³ each independently is selected from a branched (C₃-C₁₈)alkylor (C₃-C₇)cycloalkyl, or R² and R³ together with the carbon atom towhich they are attached form a fused, spiro or bridged cyclic orpolycyclic ring;

R⁵ and R⁶ each independently is selected from H, (C₁-C₁₈)alkyl,(C₂-C₁₈)alkenyl, (C₂-C₁₈)alkynyl, (C₃-C₇)cycloalkyl, or aryl, or R⁵ andR⁶ together with the oxygen atoms to which they are attached form aheterocyclic ring;

R⁷, R⁸ and R⁹ each independently is H, or an electron acceptor groupsuch as halogen, —NO₂, —CN, —COOR¹⁰, —C(═O)R¹⁰ and —SO₂R¹⁰;

R¹⁰ each independently is H or —(C₁-C₁₈)alkyl; and

Q is a protecting group such as —CH₃, —CH₂OCH₃, —C(═O)C(CH₃)₃,—CH₂—CH═CH₂, TBDMS, TBDPS, benzyl, and 2-nitro-4,5-dimethoxybenzyl.

The term “alkyl” typically means a linear or branched hydrocarbonradical having, e.g., 1-18 carbon atoms and includes methyl, ethyl,n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl,2,2-dimethylpropyl, n-hexyl, n-heptyl, n-octyl, and the like. The terms“alkenyl” and “alkynyl” typically mean linear and branched hydrocarbonradicals having, e.g., 2-18 carbon atoms and one or more double ortriple bond, respectively, and include ethenyl, propenyl, 3-buten-1-yl,2-ethenylbutyl, 3-octen-1-yl, and the like, and propynyl, 2-butyn-1-yl,3-pentyn-1-yl, and the like.

The term “alkylene” refers to a linear or branched divalent hydrocarbonradical having, e.g., 1-18 carbon atoms; and the terms “alkenylene” and“alkynylene” typically mean linear or branched divalent hydrocarbonradicals having, e.g., 2-18 carbon atoms, and one or more double ortriple bonds, respectively. Examples of alkylenes include, without beinglimited to, methylene, ethylene, propylene, butylene, 2-methylpropylene,pentylene, 2-methylbutylene, hexylene, 2-methylpentylene,3-methylpentylene, 2,3-dimethylbutylene, heptylene, octylene,n-tridecanylene, n-tetradecanylene, n-pentadecanylene, n-hexadecanylene,n-heptadecanylene, n-octadecanylene, n-nonadecanylene, icosanylene,henicosanylene, docosanylene, tricosanylene, tetracosanylene,pentacosanylene, and the like. Non-limiting examples of alkenylenesinclude 2-, 3-, 4-, 5- and 6-tridecenylene, tetradecenylenes such asmyristoleylene, 2-, 3-, 4-, 5-, 6- and 7-pentadecenylene,hexadecenylenes such as palmitoleylene, 2-, 3-, 4-, 5-, 6-, 7- and8-heptadecenylene, octadecenylenes such as oleylene, linoleylene,α-linoleylene, and the like; and non-limiting examples of alkynylenesinclude tridec-6-ynylene, undec-4-ynylene, and the like.

The term “cycloalkyl” means a mono- or bicyclic saturated hydrocarbylgroup having, e.g., 3-7 carbon atoms such as cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloheptyl, and the like, that may besubstituted, e.g., by one or more alkyl groups. The terms“cycloalkylene” and “cycloalkenylene” mean mono- or bicyclic hydrocarbylgroups having, e.g., 3-7 carbon atoms, and one or more double or triplebonds, respectively.

The term “heterocyclic ring” as used herein denotes a mono- orpoly-cyclic non-aromatic ring of, e.g., 5-12 atoms containing at leasttwo carbon atoms and at least three heteroatoms selected from sulfur,oxygen, nitrogen and boron, which may be saturated or unsaturated, i.e.,containing at least one unsaturated bond. Preferred are 5- or 6-memberedheterocyclic rings. The heterocyclic ring may be substituted at any ofthe carbon atoms of the ring, e.g., by one or more alkyl groups.Non-limiting examples of such radicals include4,5-di-tert-butyl-1,3,2-dioxaborolanyl and4,4,5,5-tetramethyl-1,3,2-dioxaborolanyl.

The term “aryl” denotes an aromatic carbocyclic group having, e.g.,6-14, carbon atoms consisting of a single ring or condensed multiplerings such as, but not limited to, phenyl, naphthyl, phenanthryl, andbiphenyl. The aryl may optionally be substituted by one or more groupseach independently selected from halogen, (C₁-C₈)alkyl, —O—(C₁-C₈)alkyl,—COO(C₁-C₈)alkyl, —CN, and —NO₂. The term “arylene-diyl” refers to adivalent radical derived from an aryl as defined herein by removal of afurther hydrogen atom from any of the ring atoms, e.g., phenylene andnaphthylene.

The term “heteroaryl” refers to a radical derived, e.g., from a5-10-membered mono- or poly-cyclic heteroaromatic ring containing one tothree, preferably 1-2, heteroatoms selected from N, O, or S. Examples ofmono-cyclic heteroaryls include, without being limited to, pyrrolyl,furyl, thienyl, thiazinyl, pyrazolyl, pyrazinyl, imidazolyl, oxazolyl,isoxazolyl, thiazolyl, isothiazolyl, pyridyl, pyrimidinyl,1,2,3-triazinyl, 1,3,4-triazinyl, and 1,3,5-triazinyl. Polycyclicheteroaryl radicals are preferably composed of two rings such as, butnot limited to, benzofuryl, isobenzofuryl, benzothienyl, indolyl,quinolinyl, isoquinolinyl, imidazo[1,2-a]pyridyl, benzimidazolyl,benzthiazolyl, benzoxazolyl, pyrido[1,2-a]pyrimidinyl and1,3-benzodioxinyl. The heteroaryl may optionally be substituted by oneor more groups each independently selected from halogen, (C₁-C₈)alkyl,—O—(C₁-C₈)alkyl, —COO(C₁-C₈)alkyl, —CN, and —NO₂. It should beunderstood that when a polycyclic heteroaryl is substituted, thesubstitution may be in any of the carbocyclic and/or heterocyclic rings.The term “heteroarylenediyl” denotes a divalent radical derived from a“heteroaryl” as defined herein by removal of a further hydrogen atomfrom any of the ring atoms.

The term “halogen” as used herein refers to a halogen and includesfluoro, chloro, bromo, and iodo, but it is preferably fluoro or chloro.

The term “amino acid” as used herein refers to an organic compoundcomprising both amine and carboxylic acid functional groups, which maybe either a natural or non-natural amino acid. The twenty two aminoacids naturally occurring in proteins are aspartic acid (Asp), tyrosine(Tyr), leucine (Leu), tryptophan (Trp), arginine (Arg), valine (Val),glutamic acid (Glu), methionine (Met), phenylalanine (Phe), serine(Ser), alanine (Ala), glutamine (Gln), glycine (Gly), proline (Pro),threonine (Thr), asparagine (Asn), lysine (Lys), histidine (His),isoleucine (Ile), cysteine (Cys), selenocysteine (Sec), and pyrrolysine(Pyl). Non-limiting examples of other amino acids include citrulline(Cit), diaminopropionic acid (Dap), diaminobutyric acid (Dab), ornithine(Orn), aminoadipic acid, β-alanine, 1-naphthylalanine,3-(1-naphthyl)alanine, 3-(2-naphthyl)alanine, γ-aminobutiric acid(GABA), 3-(aminomethyl) benzoic acid, p-ethynyl-phenylalanine,p-propargly-oxy-phenylalanine, m-ethynyl-phenylalanine,p-bromophenylalanine, p-iodophenylalanine, p-azidophenylalanine,p-acetylphenylalanine, norleucine (Nle), azidonorleucine,6-ethynyl-tryptophan, 5-ethynyl-tryptophan, 3-(6-chloroindolyl)alanine,3-(6-bromoindolyl)alanine, 3-(5-bromoindolyl)alanine, azidohomoalanine,p-chlorophenylalanine, α-aminocaprylic acid, O-methyl-L-tyrosine,N-acetylgalactosamine-α-threonine, and N-acetylgalactosamine-α-serine.

The term “peptide” refers to a short chain of amino acid monomers(residues), e.g., a chain consisting of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12 or more amino acid residues, linked by peptide bonds, i.e., thecovalent bond formed when a carboxyl group of one amino acid reacts withan amino group of another. The term “peptide moiety” as used hereinrefers to a moiety of a peptide as defined herein after removal of thehydrogen bond from a carboxylic group, i.e., either the terminal or aside chain carboxylic group, thereof. Examples of such peptide moietiesinclude, without being limited to, peptide moieties comprising the aminosequence Phe-Lys, Cit-Val, Gly-Phe-Leu-Gly, Asp-Glu-Val-Asp-, orGly-Gly-Pro-Nle, or the modified amino acid sequence carboxybenzyl (Cbz)protected-Ala-Ala-Asn-ethylenediamine.

The term “protecting group” as used herein with respect to the compoundof the formula I refers to an alcohol protecting group such as, withoutlimiting, benzoyl, benzyl, methoxymethyl ether, 3-methoxyethoxymethylether, methoxytrityl (4-methoxyphenyl) diphenylmethyl), dimethoxytrityl(bis-(4-methoxyphenyl)phenylmethyl), p-methoxybenzyl ether,methylthiomethyl ether, pivaloyl, trityl (triphenylmethyl radical),2-nitro-4,5-dimethoxybenzyl, and silyl ethers, e.g., trimethylsilyl(TMS), TBDMS, tri-iso-propylsilyloxymethyl (TOM), triisopropylsilyl(TIPS), and TBDPS ethers. Particular such protecting groups are —CH₃,—CH₂OCH₃, —C(═O)C(CH₃)₃, —CH₂—CH═CH₂, TBDMS, TBDPS, benzyl, and2-nitro-4,5-dimethoxybenzyl.

The term “electron acceptor group” as used herein refers to a group ofatoms with a high electron affinity. Non-limiting examples of suchgroups include halogen, —NO₂, —SO₂R, —CN, —C(═O)R, —C(═O)OR, andC(═O)NR₂, wherein R each independently may be, e.g., hydrogen, linear orbranched (C₁-C₁₀)alkyl, or (C₄-C₁₀)aryl. Particular such electronacceptor groups include halogen, —NO₂, —SO₂R, —CN, —C(═O)R, and—C(═O)OR, wherein R each independently is H or —(C₁-C₁₈)alkyl.

In certain embodiments, the invention provides a compound of the formulaI, wherein R¹ is a linear or branched (C₁-C₈)alkyl, preferably(C₁-C₄)alkyl, more preferably methyl or ethyl.

In certain embodiments, the invention provides a compound of the formulaI, wherein R² and R³ each independently is a branched (C₃-C₁₈)alkyl or(C₃-C₇)cycloalkyl. In other embodiments, R² and R³ each independently isa branched (C₃-C₁₈)alkyl or (C₃-C₇)cycloalkyl, and together with thecarbon atom to which they are attached form a fused, spiro or bridgedpolycyclic ring. In particular such embodiments, R² and R³ together withthe carbon atom to which they are attached form adamantyl.

In certain embodiments, the invention provides a compound of the formulaI, wherein R⁵ and R⁶ each independently is (C₁-C₈)alkyl, preferably(C₃-C₆)alkyl, more preferably isopropyl, and together with the oxygenatoms to which they are attached form a heterocyclic ring. In particularsuch embodiments, R⁵ and R⁶ each is isopropyl and together with theoxygen atoms to which they are attached form4,4,5,5-tetramethyl-1,3,2-dioxaborolanyl.

In certain embodiments, the invention provides a compound of the formulaI, wherein at least one (i.e., one, two or three) of R⁷, R⁸ and R⁹ is H,and the other of R⁷, R⁸ and R⁹ each independently is an electronacceptor group as defined above. In certain particular such embodiments,R⁷, R⁸ and R⁹ each is H. In other particular such embodiments, R⁷ is anelectron acceptor group as defined above, and R⁸ and R⁹ each is H; or R⁸is an electron acceptor group as defined above, and R⁷ and R⁹ each is H;or R⁹ is an electron acceptor group as defined above, and R⁷ and R⁸ eachis H, wherein said electron acceptor group is particularly halogen, —NO₂or —CN.

In certain embodiments, the invention provides a compound of the formulaI, wherein R¹ is a linear or branched (C₁-C₈)alkyl, preferably(C₁-C₄)alkyl, more preferably methyl or ethyl; R² and R³ eachindependently is a branched (C₃-C₁₈)alkyl or (C₃-C₇)cycloalkyl, andtogether with the carbon atom to which they are attached form a fused,spiro or bridged polycyclic ring; R⁵ and R⁶ each independently is(C₁-C₈)alkyl, preferably (C₃-C₆)alkyl, more preferably isopropyl, andtogether with the oxygen atoms to which they are attached form aheterocyclic ring; and at least one of R⁷, R⁸ and R⁹ is H, and the otherof R⁷, R⁸ and R⁹ each independently is an electron acceptor groupselected from halogen, —NO₂ or —CN. In particular such embodiments, R²and R³ together with the carbon atom to which they are attached formadamantyl; or R⁵ and R⁶ each is isopropyl and together with the oxygenatoms to which they are attached form4,4,5,5-tetramethyl-1,3,2-dioxaborolanyl. In a specific such embodiment,R¹ is methyl; R² and R³ together with the carbon atom to which they areattached form adamantly; R⁵ and R⁶ each is isopropyl and together withthe oxygen atoms to which they are attached form4,4,5,5-tetramethyl-1,3,2-dioxaborolanyl; R⁷, R⁸ and R⁹ are H; and Q isTBDMS (compound I-1).

In another aspect, the invention provides a compound of the formula IIaor IIb:

wherein

R¹ is selected from a linear or branched (C₁-C₁₈)alkyl, or(C₃-C₇)cycloalkyl;

R² and R³ each independently is selected from a branched (C₃-C₁₈)alkylor (C₃-C₇)cycloalkyl, or R² and R³ together with the carbon atom towhich they are attached form a fused, spiro or bridged cyclic orpolycyclic ring;

R⁴ is a protecting group, such as those shown in Table 1 below; Pep is apeptide moiety consisting of at least two amino acid residues and linkedvia a carboxylic group thereof;

L is absent or is a linker of the formula L1, L2 or L3, optionallysubstituted at the aromatic ring with one or more substituents eachindependently selected from (C₁-C₁₈)alkyl or (C₃-C₇)cycloalkyl, whereinM is absent or is —O— or —NH—, and the asterisk represents the point ofattachment to the group Y, provided that M is —O— or —NH— unless R₄ is4,4,5,5-tetramethyl-1,3,2-dioxaborolanyl or —B(OH)₂;

Y is absent or is —O—, provided that Y is —O— unless R⁴ is4,4,5,5-tetramethyl-1,3,2-dioxaborolanyl or —B(OH)₂, and L is absent;

R⁷, R⁸ and R⁹ each independently is H, or an electron acceptor groupsuch as halogen, —NO₂, —CN, —COOR¹⁰, —C(═O)R¹⁰ and —SO₂R¹⁰;

R¹⁰ each independently is H or —(C₁-C₁₈)alkyl;

X is a linker of the formula —X₁—X₂—, wherein X₁ is selected from(C₁-C₁₈)alkylene, (C₂-C₁₈)alkenylene, (C₂-C₁₈)alkynylene,(C₃-C₇)cycloalkylene, (C₃-C₇)cycloalkenylene, (C₆-C₁₄)arylene-diyl,(C₁-C₁₈)alkylene-(C₆-C₁₄)arylene-diyl, heteroarylenediyl, or(C₁-C₁₈)alkylene-heteroarylenediyl, said (C₁-C₁₈)alkylene,(C₂-C₁₈)alkenylene, (C₂-C₁₈)alkynylene, (C₃-C₇)cycloalkylene,(C₃-C₇)cycloalkenylene, (C₆-C₁₄)arylene-diyl, or heteroarylenediyl beingoptionally substituted by one or more groups each independently selectedfrom halogen, —COR¹⁰, —COOR¹⁰, —OCOOR¹⁰, —OCON(R¹⁰)₂, —CN, —NO₂, —SR¹⁰,—OR¹⁰, —N(R¹⁰)₂, —CON(R¹⁰)₂, —SO₂R¹⁰, —SO₃H, —S(═O)R¹⁰, (C₆-C₁₀)aryl,(C₁-C₄)alkylene-(C₆-C₁₀)aryl, heteroaryl, or (C₁-C₄)alkylene-heteroaryl,and said (C₁-C₁₈)alkylene, (C₂-C₁₈)alkenylene, or (C₂-C₁₈)alkynylenebeing further optionally interrupted by one or more identical ordifferent heteroatoms selected from S, O or N, and/or at least one groupeach independently selected from —NH—CO—, —CO—NH—, —N(C₁-C₈alkyl)-,—N(C₆-C₁₀aryl)-, (C₆-C₁₀)arylene-diyl, or heteroarylenediyl; and X₂ isabsent or is —C(O)—; and

R¹⁴ is a reactive group such as —O—(C₁-C₁₈)alkyl, —N₃, —C≡CH,N-succinimidyloxy, 3-sulfo-N-succinimidyloxy, pentafluorophenyloxy,4-nitrophenyloxy, N-imidazolyl, and N-1H-benzo[d][1,2,3]triazoloxy.

TABLE 1 Certain protecting/caging groups with respect to the compoundsof the formulas IIa/IIb, IIIa/IIIb and IVa/IVb

TBDMS 2,4-dinitrobenzene 3,4,6-trimethyl- sulfonate 2,5-dioxobenzyl

4-azidobenzyloxy 4,4,5,5-tetramethyl-1,3,2- carbonyl dioxaborolanyl

4-[4,4,5,5-tetramethyl-1,3,2- —B(OH)₂ phosphonate dioxaborolanyl]benzyl

galactosyl glucosyl glucuronyl

The term “protecting group” as used herein with respect to the compoundof the formula IIa/IIb refers to an alcohol protecting group as definedwith respect to the compound of the formula I, as well as to certaincleavable groups including enzyme cleavable groups such asmonosaccharide moieties linked through a carbon atom thereof. Thisprotecting group is further referred to herein, with respect to thecompounds of the formula IIIa/IIIb and IVa/IVb, as a “caging group”.Particular protecting/caging groups are those shown in Table 1.

The term “reactive group” as used herein with respect to the compound ofthe formula IIa/IIb refers to any group capable of reacting with afunctional group (i.e., amine, carboxylic acid, sulfhydryl, hydroxyl, oraldehyde group) of a fluorophore. Examples of such groups, withoutlimiting, include —O—(C₁-C₁₈)alkyl, —N₃, —C≡CH, N-succinimidyloxy,3-sulfo-N-succinimidyloxy, pentafluorophenyloxy, 4-nitrophenyloxy,N-imidazolyl, and N-1H-benzo[d][1,2,3] triazoloxy (see Table 2).

TABLE 2 Certain reactive groups with respect to the compound of theformula IIa/IIb

N-succinimidyloxy 3-sulfo-N- pentafluorophenyloxy succinimidyloxy

4-nitrophenyloxy N-imidazolyl N-1H-benzo[d] [1,2,3]triazoloxy

In certain embodiments, the invention provides a compound of the formulaIIa or IIb, wherein R¹ is a linear or branched (C₁-C₈)alkyl, preferably(C₁-C₄)alkyl, more preferably methyl or ethyl.

In certain embodiments, the invention provides a compound of the formulaIIa or IIb, wherein R² and R³ each independently is a branched(C₃-C₁₈)alkyl or (C₃-C₇)cycloalkyl. In other embodiments, R² and R³ eachindependently is a branched (C₃-C₁₈)alkyl or (C₃-C₇)cycloalkyl, andtogether with the carbon atom to which they are attached form a fused,spiro or bridged polycyclic ring. In particular such embodiments, R² andR³ together with the carbon atom to which they are attached formadamantyl.

In certain embodiments, the invention provides a compound of the formulaIIa or IIb, wherein at least one (i.e., one, two or three) of R⁷, R⁸ andR⁹ is H, and the other of R⁷, R⁸ and R⁹ each independently is anelectron acceptor group as defined above. In certain particular suchembodiments, R⁷, R⁸ and R⁹ each is H. In other particular suchembodiments, R⁷ is an electron acceptor group as defined above, and R⁸and R⁹ each is H; or R⁸ is an electron acceptor group as defined above,and R⁷ and R⁹ each is H; or R⁹ is an electron acceptor group as definedabove, and R⁷ and R⁸ each is H, wherein said electron acceptor group isparticularly halogen, —NO₂ or —CN.

In certain embodiments, the invention provides a compound of the formulaIIa or IIb, wherein X₁ is (C₁-C₁₈)alkylene, (C₆-C₁₄)arylene-diyl, or(C₁-C₁₈)alkylene-(C₆-C₁₄)arylene-diyl, optionally substituted by one ormore groups each independently selected from halogen, COR¹⁰, —COOR¹⁰,—OCOOR¹⁰, —OCON(R¹⁰)₂, —CN, —NO₂, —SR¹⁰, —OR¹⁰, —N(R¹⁰)₂, —CON(R¹⁰)₂,—SO₂R¹⁰, —SO₃H, —S(═O)R¹⁰, (C₆-C₁₀)aryl, (C₁-C₄)alkylene-(C₆-C₁₀)aryl,heteroaryl, or (C₁-C₄)alkylene-heteroaryl, wherein R¹⁰ is H, and said(C₁-C₁₈)alkylene being further optionally interrupted by one or moreidentical or different heteroatoms selected from S, O or N, and/or atleast one group each independently selected from —NH—CO—, —CO—NH—,—N(C₁-C₈alkyl)-, —N(C₆-C₁₀aryl)-, (C₆-C₁₀)arylene-diyl, orheteroarylenediyl; and X₂ is —C(O)—. In particular such embodiments, X₁is (C₆-C₁₄)arylene-diyl or (C₁-C₄)alkylene-(C₆-C₁₄)arylene-diyl, whereinsaid (C₆-C₁₄)arylene-diyl is, e.g., phenylene, naphthylene,phenanthrylene, or biphenylene; and X₂ is —C(O)— linked to any carbonatom of the arylene-diyl. In specific embodiments, X is the linker:

i.e., X₁ is —(CH₂)-para-phenylene and X₂ is —C(O)—.

In certain embodiments, the invention provides a compound of the formulaIIa or IIb, wherein R¹⁴ is N-succinimidyloxy, or3-sulfo-N-succinimidyloxy.

In certain embodiments, the invention provides a compound of the formulaIIa or IIb, wherein R¹ is a linear or branched (C₁-C₈)alkyl, preferably(C₁-C₄)alkyl, more preferably methyl or ethyl; R² and R³ eachindependently is selected from a branched (C₃-C₁₈)alkyl or(C₃-C₇)cycloalkyl, and together with the carbon atom to which they areattached form a fused, spiro or bridged polycyclic ring; at least one ofR⁷, R⁸ and R⁹ is H, and the other of R⁷, R⁸ and R⁹ each independently isan electron acceptor group selected from halogen, —NO₂ or —CN; X is alinker of the formula —X₁—X₂—, wherein X₁ is (C₁-C₁₈)alkylene,(C₆-C₁₄)arylene-diyl, or (C₁-C₁₈)alkylene-(C₆-C₁₄)arylene-diyl,optionally substituted by one or more groups each independently selectedfrom halogen, —COH, —COOH, —OCOOH, —OCONH₂, —CN, —NO₂, —SH, —OH, —NH₂,—CONH₂, —SO₂H, —SO₃H, —S(═O)H, (C₆-C₁₀)aryl,(C₁-C₄)alkylene-(C₆-C₁₀)aryl, heteroaryl, or (C₁-C₄)alkylene-heteroaryl,and said (C₁-C₁₈)alkylene being further optionally interrupted by one ormore identical or different heteroatoms selected from S, O or N, and/orat least one group each independently selected from —NH—CO—, —CO—NH—,—N(C₁-C₈alkyl)-, —N(C₆-C₁₀aryl)-, (C₆-C₁₀)arylene-diyl, orheteroarylenediyl; and X₂ is —C(O)—; and R¹⁴ is N-succinimidyloxy, or3-sulfo-N-succinimidyloxy.

In particular such embodiments, R² and R³ together with the carbon atomto which they are attached form adamantyl; or X is a linker of theformula —X₁—X₂—, wherein X₁ is (C₆-C₁₄)arylene-diyl or(C₁-C₄)alkylene-(C₆-C₁₄)arylene-diyl, wherein said (C₆-C₁₄)arylene-diylis phenylene, naphthylene, phenanthrylene, or biphenylene; and X₂ is—C(O)— linked to any carbon atom of the arylene-diyl. In more particularsuch embodiments, R² and R³ together with the carbon atom to which theyare attached form adamantyl; and/or X₁ is —(CH₂)-para-phenylene and X₂is —C(O)—. Specific examples of such embodiments are those wherein R¹ ismethyl; R² and R³ together with the carbon atom to which they areattached form adamantyl; X₁ is —(CH₂)-para-phenylene; and X₂ is —C(O)—,e.g., such compounds wherein at least one of R⁷, R⁸ and R⁹ is H, and theother of R⁷, R⁸ and R⁹ each independently is a halogen.

In certain embodiments, the invention provides a compound of the formulaIIa or IIb as defined in any one of the embodiments above, wherein (i) Yis —O—, L is absent or a linker of the formula L1, L2 or L3, wherein Mis —O— or —NH—, and R⁴ is a protecting group; or (ii) Y is absent, L isabsent, and R⁴ is 4,4,5,5-tetramethyl-1,3,2-dioxaborolanyl or —B(OH)₂.

In specific such embodiments, R¹ is methyl; R² and R³ together with thecarbon atom to which they are attached form adamantyl; R⁷, R⁸ and R⁹ areH; Y is —O—; L is absent; R⁴ is TBDMS; X₁ is —(CH₂)-para-phenylene; X₂is —C(O)—; and R¹⁴ is N-succinimidyloxy (compounds IIa-1 and IIb-1,Table 3).

TABLE 3 Specific compounds of the formula IIa/IIb described herein

IIa-1

IIb-1

In still another aspect, the present invention provides a conjugate ofthe formula IIIa or IIIb:

wherein

R¹ is selected from a linear or branched (C₁-C₁₈)alkyl, or(C₃-C₇)cycloalkyl;

R² and R³ each independently is selected from a branched (C₃-C₁₈)alkylor (C₃-C₇)cycloalkyl, or R² and R³ together with the carbon atom towhich they are attached form a fused, spiro or bridged cyclic orpolycyclic ring;

R⁴ is a caging group, such as those shown in Table 1;

Pep is a peptide moiety consisting of at least two amino acid residuesand linked via a carboxylic group thereof;

L is absent or is a linker of the formula L1, L2 or L3, optionallysubstituted at the aromatic ring with one or more substituents eachindependently selected from (C₁-C₁₈)alkyl or (C₃-C₇)cycloalkyl, whereinM is absent or is —O— or —NH—, and the asterisk represents the point ofattachment to the group Y, provided that M is —O— or —NH— unless R₄ is4,4,5,5-tetramethyl-1,3,2-dioxaborolanyl or —B(OH)₂;

Y is absent or is —O—, provided that Y is —O— unless R₄ is4,4,5,5-tetramethyl-1,3,2-dioxaborolanyl or —B(OH)₂, and L is absent;

R⁷, R⁸ and R⁹ each independently is H, or an electron acceptor groupsuch as halogen, —NO₂, —CN, —COOR¹⁰, —CH(═O), —C(═O)R¹⁰ and —SO₂R¹⁰;

R¹⁰ each independently is H or —(C₁-C₁₈)alkyl;

X is a linker of the formula —X₁—X₂—, wherein X₁ is selected from(C₁-C₁₈)alkylene, (C₂-C₁₈)alkenylene, (C₂-C₁₈)alkynylene,(C₃-C₇)cycloalkylene, (C₃-C₇)cycloalkenylene, (C₆-C₁₄)arylene-diyl,(C₁-C₁₈)alkylene-(C₆-C₁₄)arylene-diyl, heteroarylenediyl, or(C₁-C₁₈)alkylene-heteroarylenediyl, said (C₁-C₁₈)alkylene,(C₂-C₁₈)alkenylene, (C₂-C₁₈)alkynylene, (C₃-C₇)cycloalkylene,(C₃-C₇)cycloalkenylene, (C₆-C₁₄)arylene-diyl, or heteroarylenediyl beingoptionally substituted by one or more groups each independently selectedfrom halogen, —COR¹⁰, —COOR¹⁰, —OCOOR¹⁰, —OCON(R¹⁰)₂, —CN, —NO₂, —SR¹⁰,—OR¹⁰, —N(R¹⁰)₂, —CON(R¹⁰)₂, —SO₂R¹⁰, —SO₃H, —S(═O)R¹⁰, (C₆-C₁₀)aryl,(C₁-C₄)alkylene-(C₆-C₁₀)aryl, heteroaryl, or (C₁-C₄)alkylene-heteroaryl,and said (C₁-C₁₈)alkylene, (C₂-C₁₈)alkenylene, or (C₂-C₁₈)alkynylenebeing further optionally interrupted by one or more identical ordifferent heteroatoms selected from S, O or N, and/or at least one groupeach independently selected from —NH—CO—, —CO—NH—, —N(C₁-C₈alkyl)-,—N(C₆-C₁₀aryl)-, (C₆-C₁₀)arylene-diyl, or heteroarylenediyl; and X₂ isabsent or is —C(O)—; and

Z is a moiety of a fluorophore or a derivative thereof.

The term “fluorophore” as used herein refers to a fluorescent chemicalcompound, typically containing several combined aromatic groups, orplane or cyclic molecules having several π bonds, which can re-emitlight upon light excitation. Non-limiting categories of fluorophoresinclude fluorescein-based compounds (fluorescein analogues),rhodamine-based compounds (rhodamine analogues), coumarin-basedcompounds (coumarin analogues), cyanines such as Cy5, Cy5.5, Cy5.18,Cy7, Cy7.18, and QCy, and boron-dipyrromethene (BODIPY)-based compounds.

In certain embodiments, the invention provides a conjugate of theformula IIIa or IIIb, wherein R¹ is a linear or branched (C₁-C₈)alkyl,preferably (C₁-C₄)alkyl, more preferably methyl or ethyl.

In certain embodiments, the invention provides a conjugate of theformula IIIa or IIIb, wherein R² and R³ each independently is a branched(C₃-C₁₈)alkyl or (C₃-C₇)cycloalkyl. In other embodiments, R² and R³ eachindependently is a branched (C₃-C₁₈)alkyl or (C₃-C₇)cycloalkyl, andtogether with the carbon atom to which they are attached form a fused,spiro or bridged polycyclic ring. In particular such embodiments, R² andR³ together with the carbon atom to which they are attached formadamantyl.

In certain embodiments, the invention provides a conjugate of theformula IIIa or IIIb, wherein at least one (i.e., one, two or three) ofR⁷, R⁸ and R⁹ is H, and the other of R⁷, R⁸ and R⁹ each independently isan electron acceptor group as defined above. In certain particular suchembodiments, R⁷, R⁸ and R⁹ each is H. In other particular suchembodiments, R⁷ is an electron acceptor group as defined above, and R⁸and R⁹ each is H; or R⁸ is an electron acceptor group as defined above,and R⁷ and R⁹ each is H; or R⁹ is an electron acceptor group as definedabove, and R⁷ and R⁸ each is H, wherein said electron acceptor group isparticularly halogen, —NO₂ or —CN.

In certain embodiments, the invention provides a conjugate of theformula IIIa or IIIb, wherein X₁ is (C₁-C₁₈)alkylene,(C₆-C₁₄)arylene-diyl, or (C₁-C₁₈)alkylene-(C₆-C₁₄)arylene-diyl,optionally substituted by one or more groups each independently selectedfrom halogen, COR¹⁰, —COOR¹⁰, —OCOOR¹⁰, —OCON(R¹⁰)₂, —CN, —NO₂, —SR¹⁰,—OR¹⁰, —N(R¹⁰)₂, —CON(R¹⁰)₂, —SO₂R¹⁰, —SO₃H, —S(═O)R¹⁰, (C₆-C₁₀)aryl,(C₁-C₄)alkylene-(C₆-C₁₀)aryl, heteroaryl, or (C₁-C₄)alkylene-heteroaryl,wherein R¹⁰ is H, and said (C₁-C₁₈)alkylene being further optionallyinterrupted by one or more identical or different heteroatoms selectedfrom S, O or N, and/or at least one group each independently selectedfrom —NH—CO—, —CO—NH—, —N(C₁-C₈alkyl)-, —N(C₆-C₁₀aryl)-,(C₆-C₁₀)arylene-diyl, or heteroarylenediyl; and X₂ is —C(O)—. Inparticular such embodiments, X₁ is (C₆-C₁₄)arylene-diyl or(C₁-C₄)alkylene-(C₆-C₁₄)arylene-diyl, wherein said (C₆-C₁₄)arylene-diylis, e.g., phenylene, naphthylene, phenanthrylene, or biphenylene; and X₂is —C(O)— linked to any carbon atom of the arylene-diyl. In specificembodiments, X₁ is —(CH₂)-para-phenylene and X₂ is —C(O)—.

In certain embodiments, the invention provides a conjugate of theformula IIIa or IIIb, wherein the fluorophore Z is selected from theBODIPY derivative identified herein as Z1, the fluorescein derivativeidentified herein as Z2, the Cy5 derivative identified herein as Z3, orthe QCy derivative identified herein as Z4 (Table 4).

In certain embodiments, the invention provides a conjugate of theformula IIIa or IIIb, wherein R¹ is a linear or branched (C₁-C₈)alkyl,preferably (C₁-C₄)alkyl, more preferably methyl or ethyl; R² and R³ eachindependently is selected from a branched (C₃-C₁₈)alkyl or(C₃-C₇)cycloalkyl, and together with the carbon atom to which they areattached form a fused, spiro or bridged polycyclic ring; at least one ofR⁷, R⁸ and R⁹ is H, and the other of R⁷, R⁸ and R⁹ each independently isan electron acceptor group selected from halogen, —NO₂ or —CN; X is alinker of the formula —X₁—X₂—, wherein X₁ is (C₁-C₁₈)alkylene,(C₆-C₁₄)arylene-diyl, or (C₁-C₁₈)alkylene-(C₆-C₁₄)arylene-diyl,optionally substituted by one or more groups each independently selectedfrom halogen, —COH, —COOH, —OCOOH, —OCONH₂, —CN, —NO₂, —SH, —OH, —NH₂,—CONH₂, —SO₂H, —SO₃H, —S(═O)H, (C₆-C₁₀)aryl,(C₁-C₄)alkylene-(C₆-C₁₀)aryl, heteroaryl, or (C₁-C₄)alkylene-heteroaryl,and said (C₁-C₁₈)alkylene being further optionally interrupted by one ormore identical or different heteroatoms selected from S, O or N, and/orat least one group each independently selected from —NH—CO—, —CO—NH—,—N(C₁-C₈alkyl)-, —N(C₆-C₁₀aryl)-, (C₆-C₁₀)arylene-diyl, orheteroarylenediyl; and X₂ is —C(O)—.

TABLE 4 Fluorophore moieties identified herein as Z1-Z4

Z1

Z2

Z3

Z4

In particular such embodiments, R² and R³ together with the carbon atomto which they are attached form adamantyl; or X is a linker of theformula —X₁—X₂—, wherein X₁ is (C₆-C₁₄)arylene-diyl or(C₁-C₄)alkylene-(C₆-C₁₄)arylene-diyl, wherein said (C₆-C₁₄)arylene-diylis phenylene, naphthylene, phenanthrylene, or biphenylene; and X₂ is—C(O)— linked to any carbon atom of the arylene-diyl. In more particularsuch embodiments, R² and R³ together with the carbon atom to which theyare attached form adamantyl; and/or X₁ is —(CH₂)-para-phenylene and X₂is —C(O)—. Specific examples of such embodiments are those wherein R¹ ismethyl; R² and R³ together with the carbon atom to which they areattached form adamantyl; X₁ is —(CH₂)-para-phenylene; and X₂ is —C(O)—,e.g., such compounds wherein at least one of R⁷, R⁸ and R⁹ is H, and theother of R⁷, R⁸ and R⁹ each independently is a halogen.

TABLE 5 Specific conjugates of the formula IIIa/IIIb described herein

IIIa-1-4 (Z = Z1 to Z4)

IIIb-1-4 (Z = Z1 to Z4)

IIIa-5-8 (Z = Z1 to Z4)

IIIb-5-8 (Z = Z1 to Z4)

In certain embodiments, the invention provides a compound of the formulaIIIa or IIIb as defined in any one of the embodiments above, wherein (i)Y is —O—, L is absent or a linker of the formula L1, L2 or L3, wherein Mis —O— or —NH—, and R⁴ is a caging group; or (ii) Y is absent, L isabsent, and R⁴ is 4,4,5,5-tetramethyl-1,3,2-dioxaborolanyl or —B(OH)₂.

In specific such embodiments, R¹ is methyl; R² and R³ together with thecarbon atom to which they are attached form adamantly; X₁ is—(CH₂)-para-phenylene; X₂ is —C(O)—; Z is selected from groups Z1, Z2,Z3 or Z4; and (i) R⁷, R⁸ and R⁹ are H; Y is —O—; L is absent; and R⁴ isTBDMS (compounds IIIa-1-4 wherein Z is Z1-Z4, respectively; and IIIb-1-4wherein Z is Z1-Z4, respectively); or (ii) R⁷ is Cl; R⁸ and R⁹ are H; Yis —O—; L is absent; and R⁴ is galactosyl (compounds IIIa-5-8 wherein Zis Z1-Z4, respectively; and IIIb-5-8 wherein Z is Z1-Z4, respectively).The specific compounds disclosed herein are shown in Table 5. CompoundsIIIb-6 and IIIb-7, wherein Z is Z2 or Z3 respectively, are also referredto in Study 1 herein as Probes 2 and 3, respectively.

In yet another aspect, the present invention provides a compound of theformula IVa or IVb:

wherein

R¹ is selected from a linear or branched (C₁-C₁₈)alkyl, or(C₃-C₇)cycloalkyl;

R² and R³ each independently is selected from a branched (C₃-C₁₈)alkylor (C₃-C₇)cycloalkyl, or R² and R³ together with the carbon atom towhich they are attached form a fused, spiro or bridged cyclic orpolycyclic ring;

R⁴ is H, or a caging group such as those shown in Table 1;

Pep is a peptide moiety consisting of at least two amino acid residuesand linked via a carboxylic group thereof;

L is absent or is a linker of the formula L1, L2 or L3, optionallysubstituted at the aromatic ring with one or more substituents eachindependently selected from (C₁-C₁₈)alkyl or (C₃-C₇)cycloalkyl, whereinM is absent or is —O— or —NH—, and the asterisk represents the point ofattachment to the group Y, provided that M is —O— or —NH— unless R₄ is4,4,5,5-tetramethyl-1,3,2-dioxaborolanyl or —B(OH)₂, and when R₄ is H, Lis absent;

Y is absent or is —O—, provided that Y is —O— unless R₄ is4,4,5,5-tetramethyl-1,3,2-dioxaborolanyl or —B(OH)₂, and L is absent;

R⁷ is H, or represents at least one electron acceptor group such ashalogen, —NO₂, —CN, —COOR¹⁰, —C(═O)R¹⁰ and —SO₂R¹⁰, each independentlyattached either ortho or para to the —Y-L-R⁴ group;

R¹⁰ each independently is H or —(C₁-C₁₈)alkyl; and

A is a π* acceptor group such as —CN, or —CH═CH-E, attached either orthoor para to the —Y-L-R⁴ group, wherein E is —CN, —COOH, —COO(C₁-C₁₈)alkylsuch as —COO(C₁-C₈)alkyl or —COO(C₁-C₄)alkyl, 4-pyridinyl,methylpyridinium-4-yl, 3,3-dimethyl-3H-indolyl, or1,3,3-trimethyl-3H-indol-1-ium-2-yl.

TABLE 6 Certain π* acceptor groups with respect to compounds of theformula IVa or IVb

4-pyridinyl methylpyridinium- 3,3-dimethyl- 1,3,3-trimethyl-3H- 4-yl3H-indolyl indol-1-ium-2-yl

The term “π* acceptor group” as used herein refers to any groupcontaining a π acceptor system capable of accepting electrons.

In certain embodiments, the invention provides a compound of the formulaIVa or IVb, wherein R¹ is a linear or branched (C₁-C₈)alkyl, preferably(C₁-C₄)alkyl, more preferably methyl or ethyl.

In certain embodiments, the invention provides a compound of the formulaIVa or IVb, wherein R² and R³ each independently is a branched(C₃-C₁₈)alkyl or (C₃-C₇)cycloalkyl. In other embodiments, R² and R³ eachindependently is a branched (C₃-C₁₈)alkyl or (C₃-C₇)cycloalkyl, andtogether with the carbon atom to which they are attached form a fused,spiro or bridged polycyclic ring. In a particular such embodiment, R²and R³ together with the carbon atom to which they are attached formadamantyl.

In certain embodiments, the invention provides a compound of the formulaIVa or IVb, wherein R⁷ is H, or an electron acceptor group selected fromhalogen or —CN attached either ortho or para to the —Y-L-R⁴ group. Inparticular such embodiments, R⁷ is halogen, e.g., Cl, or —CN, attachedortho to the —Y-L-R⁴ group.

In certain embodiments, the invention provides a compound of the formulaIVa or IVb, wherein A is —CH═CH-E attached ortho to the —Y-L-R⁴ group,wherein E is —CN, —COOH, —COO(C₁-C₈)alkyl, e.g., —COO(C₁-C₄)alkyl suchas —COOCH₃, —COOC₂H₅, —COOC₃H₇, —COOCH(CH₃)₂, or —COOC(CH₃)₃,4-pyridinyl, methylpyridinium-4-yl, 3,3-dimethyl-3H-indolyl, or1,3,3-trimethyl-3H-indol-1-ium-2-yl. In particular such embodiments, Eis —CN, —COOH, —COOCH₃, —COOC₂H₅, —COOC₃H₇, —COOCH(CH₃)₂, or—COOC(CH₃)₃.

In certain embodiments, the invention provides a compound of the formulaIVa or IVb, wherein R¹ is a linear or branched (C₁-C₈)alkyl, preferably(C₁-C₄)alkyl, more preferably methyl or ethyl; R² and R³ eachindependently is selected from a branched (C₃-C₁₈)alkyl or(C₃-C₇)cycloalkyl, and together with the carbon atom to which they areattached form a fused, spiro or bridged polycyclic ring; R⁷ is H, or anelectron acceptor group selected from halogen or —CN, attached eitherortho or para to the —Y-L-R⁴ group; and A is —CH═CH-E attached ortho tothe —Y-L-R⁴ group, wherein E is —CN, —COOH, —COO(C₁-C₈)alkyl,4-pyridinyl, methylpyridinium-4-yl, 3,3-dimethyl-3H-indolyl, or1,3,3-trimethyl-3H-indol-1-ium-2-yl. Particular such embodiments arethose wherein R¹ is methyl; R² and R³ together with the carbon atom towhich they are attached form adamantyl; R⁷ is H, or is an electronacceptor group selected from halogen or —CN, attached ortho to the—Y-L-R⁴ group; and E is —CN, —COOH, or —COO(C₁-C₄)alkyl such as —COOCH₃,—COOC₂H₅, —COOC₃H₇, —COOCH(CH₃)₂, or —COOC(CH₃)₃. More particular suchembodiments are those wherein E is —CN, —COOH, —COOCH₃, or —COOC(CH₃)₃,i.e., A is acrylonitrile, acrylic acid, methylacrylate or tert-butylacrylate substituent, respectively, attached ortho to the —Y-L-R⁴ group.

In certain embodiments, the invention provides a compound of the formulaIVa or IVb as defined in any one of the embodiments above, wherein (i) Yis —O—; L is absent; and R⁴ is H; (ii) Y is —O—; L is either absent or alinker of the formula L1, L2 or L3 as defined above, wherein M is —O— or—NH—; and R⁴ is a caging group such as those shown in Table 1, e.g.,phosphonate, but provided that said caging group is not4,4,5,5-tetramethyl-1,3,2-dioxaborolanyl or —B(OH)₂; (iii) Y is —O—; Lis a linker of the formula L1, L2 or L3 as defined above, wherein M isabsent; and R⁴ is 4,4,5,5-tetramethyl-1,3,2-dioxaborolanyl or —B(OH)₂;or (iv) Y is absent; L is absent; and R⁴ is4,4,5,5-tetramethyl-1,3,2-dioxaborolanyl or —B(OH)₂.

In specific such embodiments, the compound disclosed herein is acompound of the formula IVa or IVb, wherein R¹ is methyl; R² and R³together with the carbon atom to which they are attached form adamantyl;R⁷ is H, or Cl attached ortho to the —Y-L-R⁴ group; A is —CH═CH-Eattached ortho to the —Y-L-R⁴ group; and (i) E is —COOC(CH₃)₃; Y is —O—;L is absent; and R⁴ is galactosyl (e.g., compounds IVa-1, IVa-2, IVb-1and IVb-2); (ii) E is —COOCH₃ or —CN; Y is —O—; L is absent; and R⁴ is H(e.g., compounds IVa-3, IVa-4, IVa-5, IVa-6, IVb-3, IVb-4, IVb-5 andIVb-6); (iii) E is —COOCH₃ or —CN; Y is —O—; L is L1 wherein M is —O—;and R⁴ is galactosyl (e.g., compounds IVa-7, IVa-8, IVa-9, IVa-10,IVb-7, IVb-8, IVb-9 and IVb-10); (iv) E is —COOCH₃; Y is —O—; L is L1wherein M is —NH—; and R⁴ is 2,4-dinitrobenzene sulfonate (e.g.,compounds IVa-11, IVa-12, IVb-11 and IVb-12); (v) E is —COOH; Y isabsent; L is absent; and R⁴ is 4,4,5,5-tetramethyl-1,3,2-dioxaborolanyl(e.g., compounds IVa-13, IVa-14, IVb-13 and IVb-14); or (vi) E is —COOH;Y is —O—; L is absent; and R⁴ is phosphonate (e.g., compounds IVa-15,IVa-16, IVb-15 and IVb-16). The specific compounds disclosed herein areshown in Table 7. Compounds IVb-7, IVb-8, IVb-9, IVb-10, IVb-13, IVb-15and IVb-11 are also referred to in Study 2 herein as Probes 6, 7, 8, 9,10, 11 and 12, respectively.

TABLE 7 Specific compounds of the formula IVa/IVb described herein

IVa-1

IVb-1

IVa-2

IVb-2

IVa-3

IVb-3

IVa-4

IVb-4

IVa-5

IVb-5

IVa-6

IVb-6

IVa-7

IVb-7

IVa-8

IVb-8

IVa-9

IVb-9

IVa-10

IVb-10

IVa-11

IVb-11

IVa-12

IVb-12

IVa-13

IVb-13

IVa-14

IVb-14

IVa-15

IVb-15

IVa-16

IVb-16

In a further aspect, the present invention provides a compositioncomprising a carrier, and a dioxetane-based chemiluminescence probe asdisclosed herein, i.e., either a fluorophore-tethered dioxetane-basedchemiluminescence probe of the formula IIIa/IIIb or a π* acceptorgroup-containing chemiluminescence probe of the formula IVa/IVb, each asdefined in any one of the embodiments above.

In specific embodiments, the composition of the present inventioncomprises a chemiluminescence probe of the formula IIIa/IIIb selectedfrom those listed in Table 5, or a chemiluminescence probe of theformula IVa/IVb selected from those listed in Table 7.

As shown herein, the composition of the present invention may thus beused for diagnostics and/or in vivo imaging. Triggered chemiluminescenceemission can provide a highly sensitive readout of biological analytes.Chemiluminescence does not require light excitation, thereby drasticallyreducing background from autofluorescence and photoactivation offunctional groups. Whereas bioluminescence, i.e., chemiluminescencederived from living systems that express bioluminescent enzymes such asluciferase, has found wide application for preclinical analysis ofbiological parameters using genetically modified organisms, smallmolecule chemiluminescence can be used with wild-type animals and opensup exciting opportunities for clinical imaging.

The compositions of the present invention may thus be inter aliapharmaceutical compositions, wherein said carrier is a pharmaceuticallyacceptable carrier.

As described above, chemiluminescence probes of the formulas IIIa/IIIband IVa/IVb as disclosed herein have a cleavable caging group (R⁴),e.g., an enzyme cleavable group, wherein removal of said cleavable groupby the analyte of interest, e.g., in the presence of an enzyme capableof cleaving said enzyme cleavable group, generates an unstablephenolate-dioxetane species that decomposes through a chemiexcitationprocess to produce the excited intermediate, which then further decaysto its ground-state through emission of light.

Particular chemiluminescence probes exemplified herein, havingβ-galactosyl as the caging group, are the fluorophore-tethereddioxetane-based chemiluminescence Probes 2 and 3, and the π* acceptorgroup-containing chemiluminescence Probes 6-9, and theirchemiluminescent kinetic profiles in the presence vs. absent ofβ-galactosidase is shown in Studies 1 and 2. Additional probesexemplified in Study 2 are the π* acceptor group-containingchemiluminescence Probes 11 and 12, having phosphonate or2,4-dinitrobenzene sulfonate as the caging group, which are capable ofdetecting alkaline-phosphatase and GSH, respectively; and the π*acceptor group-containing chemiluminescence Probe 10, having4,4,5,5-tetramethyl-1,3,2-dioxaborolanyl as the caging group, which iscapable of detecting hydrogen peroxide.

Other chemiluminescence probes may have a caging group comprising apeptide moiety consisting of two or more amino acid residues, e.g., apeptide moiety-containing caging group as shown in Table 1. Such peptidemoieties may comprise an amino acid sequence cleavable by a specificenzyme, and probes containing such caging groups may thus be used fordetecting the presence of said enzyme.

One particular such enzyme is cathepsin B, a lysosomal cysteine proteasethat plays an important role in intracellular proteolysis and isoverexpressed in premalignant lesions and various pathologicalconditions, as well as in cancers, e.g., in tumor endothelial cells andmany other tumor cells in the lysosome (Miller et al., 2009). CathepsinB-cleavable peptides include, without limiting, peptides comprising theamino acid sequence Phe-Lys, Cit-Val, or Gly-Phe-Leu-Gly. Anotherparticular such enzyme is cathepsin K, a lysosomal cysteine proteaseinvolved in bone remodeling and resorption, which is expressedpredominantly in osteoclasts and overexpressed extracellularly in boneneoplasms (Segal et al., 2009). Cathepsin K-cleavable peptides include,without being limited to, peptides comprising the amino acid sequenceGly-Gly-Pro-Nle. A further particular such enzyme is legumain, alysosomal enzyme overexpressed in tumor cells (Stern et al., 2009).Legumain-cleavable peptides include, without limiting, peptidescomprising the modified amino acid sequenceCbz-Ala-Ala-Asn-ethylenediamine.

Pharmaceutical compositions according to the present invention may beprepared by conventional techniques, e.g., as described in Remington:The Science and Practice of Pharmacy, 19^(th) Ed., 1995. Thecompositions can be prepared, e.g., by uniformly and intimately bringingthe active agent, i.e., the dioxetane-based chemiluminescence probe,into association with a liquid carrier, a finely divided solid carrier,or both, and then, if necessary, shaping the product into the desiredformulation. The compositions may be in liquid, solid or semisolid formand may further include pharmaceutically acceptable fillers, carriers,diluents or adjuvants, and other inert ingredients and excipients. Inone embodiment, the pharmaceutical composition of the present inventionis formulated as nanoparticles.

A pharmaceutical composition according to the present invention can beformulated for any suitable route of administration, e.g., forparenteral administration such as intravenous, intraarterial,intrathecal, intrapleural, intratracheal, intraperitoneal, intramuscularor subcutaneous administration, topical administration, oral or enteraladministration, or for inhalation. In particular embodiments, such acomposition is formulated for intravenous or intraperitonealadministration, or for subcutaneous administration, e.g., by an alzetpump implanted subcutaneous.

The pharmaceutical composition of the invention may be in the form of asterile injectable aqueous or oleaginous suspension, which may beformulated according to the known art using suitable dispersing, wettingor suspending agents. The sterile injectable preparation may also be asterile injectable solution or suspension in a non-toxic parenterallyacceptable diluent or solvent. Acceptable vehicles and solvents that maybe employed include, e.g., water, Ringer's solution and isotonic sodiumchloride solution.

The chemiluminescence emission of the probes of the present inventioncan be detected utilizing any technique or procedure known in the art.

Optical molecular imaging is a promising technique that provides a highdegree of sensitivity and specificity in tumor margin detection.Furthermore, existing clinical applications have proven that opticalmolecular imaging is a powerful intraoperative tool for guiding surgeonsperforming precision procedures, thus enabling radical resection andimproved survival rates. An example of a clinically approved instrumentfor minimally invasive surgical procedures under fluorescence guidanceis the da Vinci Surgical System (Haber et al., 2010). This instrument isfeatured with a 3D HD vision system for a clear and magnified viewinside a patient's body and allows surgeons to perform complex androutine procedures through a few small openings, similar to traditionallaparoscopy. In addition, the following systems have already beenapplied in surgeries for breast cancer, liver metastases and bypassinggraft surgery: The Hamamatsu's Photodynamic Eye (PDE™), Artemis™ andNovadaq SPY™ (Novadaq Technologies Inc., Toronto, Canada) (Chi et al.,2014). Several existing intraoperative NIR fluorescence molecularimaging systems were evaluated in clinical trials; including, Fluobeam®,FLARE™ and GXMI Navigator. They have played an important role inoperation convenience, improving image assessment and increasingdetection depth (Chi et al., 2014).

In recent years, there has been a great progress in the development ofcameras and lasers for optical fluorescence imaging in the IR range(Mieog et al., 2011; Troyan et al., 2009). In parallel, there is a vastclinical use of low MW organic dyes such as ICG and methylene blue fordetermining cardiac output, hepatic function and liver blood flow, andfor ophthalmic angiography. In 2015, the fluorescence imaging system,Xiralite®, gained FDA approval for visualization of microcirculation inthe hands (for inflammation and perfusion-related disorders).

The invention will now be illustrated by the following non-limitingExamples.

EXAMPLES

Study 1. Remarkable Enhancement of Chemiluminescent Signal byDioxetane-Fluorophore Conjugates: Turn-ON Chemiluminescence Probes withColor Modulation for Sensing and Imaging

Experimental

General.

All reactions requiring anhydrous conditions were performed under anargon atmosphere. All reactions were carried out at RT unless statedotherwise. Chemicals and solvents were either analytical reagents (A.R.)grade or purified by standard techniques. TLC: silica gel plates Merck60 F254: compounds were visualized by irradiation with UV light. Flashchromatography (FC): silica gel Merck 60 (particle size 0.040-0.063 mm),eluent given in parentheses. RP-HPLC: C18 5 u, 250×4.6 mm, eluent givenin parentheses. Preparative RP-HPLC: C18 5 u, 250×21 mm, eluent given inparentheses. ¹H-NMR spectra were recorded using Bruker Avance operatedat 400 MHz. ¹³C-NMR spectra were recorded using Bruker Avance operatedat 100 MHz. Chemical shifts were reported in ppm on the δ scale relativeto a residual solvent (CDCl₃: δ=7.26 for ¹H-NMR and 77.16 for ¹³C-NMR,DMSO-d₆: δ=2.50 for ¹H-NMR and 39.52 for ¹³C-NMR). Mass spectra weremeasured on Waters Xevo TQD. Fluorescence and chemiluminescence wererecorded on Molecular Devices Spectramax i3x. Images of microplate andmice were recorded on BioSpace Lab PhotonIMAGER™. All reagents,including salts and solvents, were purchased from Sigma-Aldrich.

Compound 1b.

2-chloro-3-hydroxybenzaldehyde (1a, 2000 mg, 12.77 mmol) was dissolvedin 20 ml of methanol. Trimethyl orthoformate (2.24 ml, 20.44 mmol) andtertrabutylammonium tribromide (308 mg, 0.64 mmol) were added and thesolution was stirred at RT. Reaction was monitored by TLC. Uponcompletion, reaction mixture was diluted with EtOAc (100 ml) and washedwith 0.01M NaHCO₃ (100 ml). Organic phase was dried over Na₂SO₄ andconcentrated under reduced pressure. Purification by columnchromatography (Hex:EtOAc 80:20) afforded 2460 mg (95% yield) ofcolorless oil. ¹H NMR (400 MHz, CDCl₃): δ 7.22-7.17 (m, 2H), 7.04-6.99(m, 1H), 5.82 (s, 1H), 5.58 (s, 1H), 3.37 (s, 6H). ¹³C NMR (100 MHz,CDCl₃): δ 151.65, 135.87, 127.58, 119.90, 119.13, 116.42, 101.03, 53.77.MS (ES−): m/z calc. for C₉H₁₁ClO₃: 202.0; found: 201.1 [M−H]⁻.

Compound 1c.

Phenol 1b (2450 mg, 12.09 mmol) and imidazole (1650 mg, 24.24 mmol) weredissolved in 15 ml of DCM. TBSCl (2180 mg, 14.46 mmol) was added and thesolution was stirred at RT. Reaction was monitored by TLC. Uponcompletion, the white precipitate was filtered-off and the solvent wasevaporated under reduced pressure. Purification by column chromatography(Hex:EtOAc 95:5) afforded 3600 mg (94% yield) of colorless oil. ¹H NMR(400 MHz, CDCl₃): δ 7.23 (dd, J=7.8, 1.5 Hz, 1H), 7.14 (t, J=7.9 Hz,1H), 6.88 (dd, J=8.0, 1.5 Hz, 1H), 5.63 (s, 1H), 3.37 (s, 6H), 1.03 (s,9H), 0.22 (s, 6H). ¹³C NMR (100 MHz, CDCl₃): δ 151.81, 137.01, 126.74,125.01, 120.62, 120.50, 101.29, 53.93, 25.81, 18.48, −4.23. MS (ES+):m/z calc. for C₁₅H₂₅ClO₃Si: 316.1; found: 285.1 [M-CH₃O⁻]⁺.

Compound 1d.

Acetal 1c (3500 mg, 11.04 mmol) and trimethyl phosphite (1.7 ml, 14.41mmol) were dissolved in 30 ml of DCM. Reaction mixture was cooled to 0°C. and titanium(IV) chloride (1.45 ml, 13.22 mmol) was added dropwise.Reaction was monitored by TLC. Upon completion, the solution was pouredinto a saturated aqueous solution of NaHCO₃ (130 ml) at 0° C. After 10minutes of stirring, 100 ml of DCM was added and the phases wereseparated. Organic phase was dried over Na₂SO₄ and concentrated underreduced pressure. Purification by column chromatography (Hex:EtOAc40:60) afforded 4010 mg (92% yield) of colorless oil. ¹H NMR (400 MHz,CDCl₃): δ 7.27 (dt, J=7.8, 1.9 Hz, 1H), 7.19 (t, J=7.9 Hz, 1H), 6.88(dt, J=7.9, 1.6 Hz, 1H), 5.19 (d, J=15.7 Hz, 1H), 3.78 (d, J=10.6 Hz,3H), 3.64 (d, J=10.5 Hz, 3H), 3.35 (s, 3H), 1.02 (s, 9H), 0.22 (s, 6H).¹³C NMR (100 MHz, CDCl₃): δ 151.71, 134.03, 127.28, 126.10, 121.89,120.48, 77.30, 75.60, 58.83, 53.81, 25.77, 18.46, −4.29. MS (ES+): m/zcalc. for C₁₆H₂₈ClO₅PSi: 394.1; found: 395.3 [M+H]⁺.

Compound 1e.

Phosphonate 1d (3950 mg, 10.0 mmol) was dissolved in 25 ml of anhydrousTHF under argon atmosphere at −78° C. LDA (2.0 M in THF, 6 ml, 12 mmol)was added and the solution was stirred for 20 minutes. A solution of2-adamantanone (2250 mg, 14.98 mmol) in 20 ml of THF was added, andafter 15 minutes of stirring at −78° C. reaction was allowed to warm toRT. Reaction was monitored by TLC. Upon completion, reaction mixture wasdiluted with EtOAc (150 ml) and washed with brine (150 ml). Organicphase was dried over Na₂SO₄ and concentrated under reduced pressure.Purification by column chromatography (Hex:EtOAc 95:5) afforded 3560 mg(85% yield) of white solid. ¹H NMR (400 MHz, CDCl₃): δ 7.09 (t, J=8.0Hz, 1H), 6.89-6.84 (m, 2H), 3.30 (s, 3H), 3.27 (s, 1H), 2.05 (s, 1H),1.97-1.65 (m, 12H), 1.04 (s, 9H), 0.23 (s, 6H). ¹³C NMR (100 MHz,CDCl₃): δ 151.98, 140.24, 136.20, 130.69, 126.69, 126.58, 124.93,120.30, 56.98, 39.28, 39.14, 38.74, 37.32, 32.96, 29.70, 28.58, 28.43,25.86, 18.54, −4.28. MS (ES+): m/z calc. for C₂₄H₃₅ClO₂Si: 418.2; found:419.3 [M+H]⁺.

Compound 1f.

Compound 1e (3500 mg, 8.35 mmol) was dissolved in 30 ml of THF.Tetrabutylammonium fluoride (1.0 M in THF, 9.2 ml, 9.2 mmol) was addedand the solution was stirred at RT. Reaction was monitored by TLC. Uponcompletion, reaction mixture was diluted with EtOAc (150 ml) and washedwith 1M HCl (100 ml). Organic phase was dried over Na₂SO₄ andconcentrated under reduced pressure. Purification by columnchromatography (Hex:EtOAc 85:15) afforded 2420 mg (95% yield) of whitesolid. ¹H NMR (400 MHz, CDCl₃): δ 7.15 (t, J=7.8 Hz, 1H), 6.99 (dd,J=8.2, 1.3 Hz, 1H), 6.83 (dd, J=7.5, 1.3 Hz, 1H), 5.90 (s, 1H), 3.31 (s,3H), 3.27 (s, 1H), 2.10 (s, 1H), 2.00-1.64 (m, 12H). ¹³C NMR (100 MHz,CDCl₃): δ 151.82, 139.77, 135.06, 131.96, 127.39, 123.95, 120.68,115.60, 57.16, 39.23, 39.14, 38.85, 38.71, 37.18, 32.89, 29.73, 28.46,28.34. MS (ES−): m/z calc. for C₁₈H₂₁ClO₂: 304.1; found: 303.2 [M−H]⁻.

Compound 1g.

Phenol 1f (1500 mg, 4.92 mmol) was dissolved in 5 ml of DCM and 10 ml ofquinoline. Acetobromo-α-D-galactose (2430 mg, 5.91 mmol) and silvercarbonate (1760 mg, 6.38 mmol) were added and the solution was stirredat RT. Reaction was monitored by TLC. Upon completion, reaction mixturewas diluted with DCM (120 ml) followed by filtration over celite. Thefiltrate was washed with 1M HCl (2×100 ml) and brine (100 ml). Organicphase was dried over Na₂SO₄ and concentrated under reduced pressure.Purification by column chromatography (Hex:EtOAc 70:30) afforded 2720 mg(87% yield) of white solid. ¹H NMR (400 MHz, CDCl₃): δ 7.17 (d, J=5.1Hz, 2H), 7.01 (t, J=4.5 Hz, 1H), 5.59 (t, J=9.2 Hz, 1H), 5.47 (dd,J=3.3, 0.7 Hz, 1H), 5.11 (dd, J=10.5, 3.4 Hz, 1H), 4.98 (dd, J=8.0, 2.2Hz, 1H), 4.31-4.23 (m, 1H), 4.20-4.13 (m, 1H), 4.08-4.03 (m, 1H), 3.33(s, 1H), 3.26 (s, 3H), 2.19 (s, 3H), 2.08 (s, 3H), 2.07 (s, 3H), 2.01(s, 3H), 2.00-1.66 (m, 13H). ¹³C NMR (100 MHz, CDCl₃): δ 170.51, 170.39,170.32, 169.57, 152.99, 139.74, 136.42, 131.54, 127.08, 126.82, 125.32,118.07, 100.97, 71.24, 70.77, 68.32, 66.95, 61.45, 57.45, 57.07, 39.29,39.19, 38.84, 38.68, 37.19, 32.88, 29.78, 28.46, 28.30, 20.99, 20.80,20.73. MS (ES+): m/z calc. for C₃₂H₃₉ClO₁₁: 634.2; found: 635.3 [M+H]⁺.

Compound 1h.

Compound 1g (2000 mg, 3.15 mmol), bis(pinacolato)diboron (1440 mg, 5.67mmol), (1,5-cyclooctadiene)(methoxy)iridium(I) dimer (42 mg, 0.063 mmol)and BBBPY (34 mg, 0.127 mmol) were dissolved in 20 ml of anhydrous THFin a sealed tube. Reaction mixture was stirred at 80° C. for 2 hours,and was monitored by ¹H-NMR (appearance of two aromatic hydrogens at7.48 and 7.43 ppm and disappearance of the aromatic hydrogens of 1g).Upon completion, the solvent was evaporated under reduced pressure. Thecrude product was passed through silica gel column (Hex:EtOAc 65:35) toafford 2130 mg (89% yield) of white solid that was taken to the nextstep without further purification.

Compound 1j.

Arylboronate ester 1h (2100 mg, 2.76 mmol), benzyl bromide 1i (Jacobsonet al., 1988) (950 mg, 3.04 mmol) and potassium carbonate (950 mg, 6.87mmol) were dissolved in 20 ml of anhydrous 1,4-dioxane. The solution wasthoroughly degassed by bubbling of argon and thentetrakis(triphenylphosphine)palladium(0) (320 mg, 0.28 mmol) was added.The flask was sealed and the solution was stirred at 120° C. for 2hours. Reaction was monitored by TLC. Upon completion, the solvent wasevaporated under reduced pressure. Purification by column chromatography(Hex:EtOAc 40:60) afforded 950 mg (40% yield) of white solid. ¹H NMR(400 MHz, CDCl₃): δ 8.06 (d, J=8.3 Hz, 2H), 7.30 (d, J=8.3 Hz, 2H), 7.00(s, 1H), 6.82 (s, 1H), 5.55 (t, J=9.2 Hz, 1H), 5.44 (d, J=2.8 Hz, 1H),5.09 (dd, J=10.5, 3.4 Hz, 1H), 4.94 (dd, J=7.8, 2.6 Hz, 1H), 4.19-4.13(m, 2H), 4.06-3.96 (m, 3H), 3.31 (s, 1H), 3.24 (s, 3H), 2.89 (s, 4H),2.17 (s, 3H), 2.07 (s, 3H), 2.02 (s, 3H), 2.00 (s, 3H), 1.98-1.63 (m,13H). ¹³C NMR (100 MHz, CDCl₃): δ 170.42, 170.34, 170.24, 169.54,169.38, 161.73, 152.93, 147.95, 138.59, 136.73, 136.51, 131.10, 129.31,127.73, 127.52, 123.57, 123.50, 118.93, 100.88, 71.18, 70.66, 68.28,66.89, 61.37, 57.52, 57.14, 41.58, 39.17, 39.09, 38.82, 38.61, 37.13,32.99, 32.94, 29.74, 29.69, 28.38, 28.28, 25.77, 24.93, 24.67, 20.96,20.75, 20.69. MS (ES+): m/z calc. for C₄₄H₄₈ClNO₁₅: 865.3; found: 888.4[M+Na]⁺.

Compound 1k.

Enol ether 1j (250 mg, 0.29 mmol) and few milligrams of methylene bluewere dissolved in 20 ml of DCM. Oxygen was bubbled through the solutionwhile irradiating with yellow light. Reaction was monitored by TLC. Uponcompletion, the solvent was concentrated under reduced pressure.Purification by column chromatography (Hex:EtOAc 35:65) afforded 238 mg(92% yield) of white solid. The product was isolated as a mixture ofdiastereomers. ¹H NMR (400 MHz, CDCl₃): δ 8.06 (d, J=7.9 Hz, 2H),7.75-7.67 (m, 1H), 7.31 (d, J=7.5 Hz, 2H), 7.12-7.02 (m, 1H), 5.59-5.49(m, 1H), 5.42 (s, 1H), 5.07 (d, J=10.3 Hz, 1H), 4.87 (d, J=7.8 Hz, 1H),4.14-4.10 (m, 2H), 4.08 (s, 2H), 4.03-3.95 (m, 1H), 3.22-3.10 (m, 3H),3.02-2.94 (m, 1H), 2.88 (s, 4H), 2.32-2.21 (m, 1H), 2.15 (s, 3H),2.07-1.95 (m, 9H), 1.93-1.53 (m, 12H). ¹³C NMR (100 MHz, CDCl₃): δ170.32, 170.22, 170.11, 169.41, 169.32, 161.66, 153.87, 147.63, 139.21,133.92, 131.12, 129.29, 128.62, 123.61, 120.10, 111.81, 100.95, 96.42,71.28, 70.54, 68.32, 68.22, 66.84, 66.79, 61.31, 61.23, 49.75, 41.75,36.60, 33.84, 33.73, 33.64, 32.67, 32.31, 31.63, 31.52, 26.21, 26.00,25.75, 22.70, 20.90, 20.68, 20.62, 14.17. MS (ES+): m/z calc. forC₄₄H₄₈ClNO₁₇: 897.3; found: 920.7 [M+Na]⁺.

Probe 1.

Enol ether 1g (100 mg, 0.157 mmol) and few milligrams of methylene bluewere dissolved in 10 ml of DCM. Oxygen was bubbled through the solutionwhile irradiating with yellow light. Reaction was monitored by TLC. Uponcompletion, the solvent was concentrated under reduced pressure and thecrude product was passed through silica gel column (Hex:EtOAc 60:40) toremove methylene blue. The solvent was evaporated and the product wasdissolved in MeOH (3 ml). Potassium carbonate (87 mg, 0.63 mmol) wasadded and the solution was stirred at RT. Reaction was monitored byRP-HPLC. Purification by RP-HPLC (30-100% ACN in water, 20 min) afforded67 mg (85% yield) of white solid. The product was isolated as a mixtureof diastereomers. ¹H NMR (400 MHz, CDCl₃): δ 7.79 (d, J=7.3 Hz, 1H),7.30 (t, J=7.8 Hz, 1H), 7.22 (d, J=8.1 Hz, 1H), 4.89-4.72 (m, 1H),4.34-4.19 (m, 5H), 4.15-4.07 (m, 1H), 3.91-3.77 (m, 3H), 3.64 (m, 1H),3.20-3.04 (m, 3H), 2.96 (s, 1H), 2.28-2.17 (m, 1H), 2.01-1.46 (m, 12H).¹³C NMR (100 MHz, CDCl₃): δ 153.52, 133.82, 127.55, 122.27, 118.89,111.91, 102.45, 96.22, 74.51, 73.24, 71.20, 69.06, 61.46, 49.73, 36.66,34.01, 33.57, 32.71, 32.30, 31.72, 26.20, 25.97, 22.79, 14.26. MS (ES−):m/z calc. for C₂₄H₃₁ClO₉: 498.2; found: 543.3 [M+HCOO⁻]⁻.

Compound 2b.

Fluorescein isothiocyanate 2a (150 mg, 0.385 mmol) andN-Boc-ethylenediamine (68 mg, 0.42 mmol) were dissolved in 2 ml of DMF.3 drops of Et₃N were added and the solution was stirred at RT. Reactionwas monitored by TLC. Upon completion the solvent was evaporated underreduced pressure. Purification by column chromatography (Hex:EtOAc25:75) afforded 192 mg (91% yield) of orange solid. ¹H NMR (400 MHz,DMSO): δ 10.45-9.76 (m, 3H), 8.21 (s, 1H), 8.05 (s, 1H), 7.74 (d, J=6.4Hz, 1H), 7.18 (d, J=8.2 Hz, 1H), 6.95 (s, 1H), 6.68 (s, 2H), 6.63-6.53(m, 4H), 3.48-3.35 (m, 2H), 3.22-3.09 (m, 2H), 1.38 (s, 9H). ¹³C NMR(100 MHz, DMSO): δ 180.70, 168.54, 159.54, 155.87, 151.92, 147.30,141.19, 129.68, 129.07, 126.60, 124.13, 116.72, 112.63, 109.74, 102.28,77.86, 43.83, (two peaks of ethylenediamine linker are hidden undersolvent signal), 28.26. MS (ES+): m/z calc. for C₂₈H₂₇N₃O₇S: 549.2;found: 550.3 [M+H]⁺.

Compound 2c.

Compound 2b (40 mg, 0.073 mmol) was dissolved in 2 ml of 1:1 mixture ofTFA and DCM. Reaction was stirred at RT and monitored by TLC. Uponcompletion, the solvent was removed under reduced pressure and theproduct was taken to the next step without further purification.

Probe 2.

Amine-functionalized fluorescein 2c (41 mg, 0.073 mmol) and NHS ester 1k(65.5 mg, 0.073 mmol) were dissolved in 1 ml of DMF. The flask was keptin the dark by covering it with an aluminum foil and 2 drops of Et₃Nwere added. Reaction was stirred at RT and was monitored by RP-HPLC.Upon completion, the solvent was evaporated under reduced pressure andthe resulting yellow solid was dissolved in 1.5 ml of MeOH. Potassiumcarbonate (40 mg, 0.29 mmol) was added and removal of sugar acetates wasmonitored by RP-HPLC. Upon completion the product was purified byRP-HPLC (30-100% ACN in water, 20 min) to afford 57 mg (74% yield) ofyellow solid. The product was isolated as a mixture of diastereomers. ¹HNMR (400 MHz, DMSO): δ 10.33-9.85 (m, 3H), 8.59 (s, 1H), 8.22 (d, J=1.7Hz, 1H), 8.16 (s, 1H), 7.81 (d, J=7.6 Hz, 2H), 7.76-7.69 (m, 1H), 7.45(s, 1H), 7.37-7.28 (m, 3H), 7.17 (d, J=8.3 Hz, 1H), 6.67 (d, J=2.3 Hz,2H), 6.63-6.52 (m, 4H), 5.04-4.85 (m, 1H), 4.21-3.94 (m, 5H=2H ofbenzylic singlet+3H of the sugar moiety, under broad H₂O peak),3.78-3.38 (m, 11H), 3.08-2.99 (m, 3H), 2.84 (s, 1H), 2.32-2.16 (m, 1H),1.94-1.35 (m, 12H). ¹³C NMR (100 MHz, DMSO): δ 180.74, 168.45, 166.56,159.48, 153.62, 153.38, 151.88, 147.29, 143.99, 141.15, 140.26, 132.33,132.03, 129.77, 129.31, 129.01, 128.49, 127.54, 126.57, 125.51, 124.07,118.17, 118.03, 117.87, 116.88, 112.57, 111.51, 109.72, 102.23, 101.05,100.38, 95.27, 75.59, 73.54, 70.22, 70.13, 67.97, 60.13, 59.74, 51.43,49.22, 43.57, 38.46, 35.89, 33.21, 32.89, 31.91, 31.75, 31.05, 30.75,28.99, 28.24, 26.83, 25.49, 25.16. MS (ES−): m/z calc. forC₅₅H₅₄ClN₃O₁₅S: 1063.3; found: 1062.7 [M−H]⁻.

Compound 3b.

Compound 3a (Karton-Lifshin et al., 2012) (180 mg, 0.30 mmol),N-Boc-ethylenediamine (96 mg, 0.60 mmol) and2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU) (228 mg, 0.60 mmol) were dissolved in 3 ml of DMF. Triethylamine(120 μL, 0.86 mmol) was added and reaction mixture was stirred at RT.Reaction was monitored by RP-HPLC (10-90% ACN in water, 20 min). Uponcompletion, the solvent was evaporated under reduced pressure. Crudeproduct was dissolved in 5 ml of H₂O, few drops of MeOH and few drops ofAcOH. Purification by RP-HPLC (10-90% ACN in water, 20 min) afforded 187mg (84% yield) of yellow solid. ¹H NMR (400 MHz, DMSO): δ 8.88 (d, J=6.7Hz, 4H), 8.69 (s, 1H), 8.33 (s, 2H), 8.31-8.19 (m, 6H), 7.53 (d, J=16.2Hz, 2H), 6.96 (s, 1H), 4.28 (s, 6H), 3.35 (dd, J=11.9, 6.0 Hz, 2H), 3.15(dd, J=11.9, 5.9 Hz, 2H), 1.37 (s, 9H). ¹³C NMR (100 MHz, DMSO): δ165.45, 157.39, 155.83, 152.51, 145.25, 135.09, 128.47, 126.54, 124.30,124.06, 123.69, 77.75, 46.97 (two peaks of ethylenediamine linker arehidden under solvent signal), 28.26 (TFA signals are not shown). MS(ES+): m/z calc. for C₃₀H₃₅N₄O₄ ⁺ (quinone form): 515.3; found: 515.5.

Compound 3c.

Compound 3b (60 mg, 0.081 mmol) was dissolved in 2 ml of 1:1 mixture ofTFA and DCM. Reaction was stirred at RT and monitored by RP-HPLC (10-90%ACN in water, 20 min). Upon completion, the solvent was removed underreduced pressure and the product was taken to the next step withoutfurther purification.

Probe 3.

Amine-functionalized QCy 3c (60 mg, 0.081 mmol) and NHS ester 1k (72.5mg, 0.081 mmol) were dissolved in 1 ml of DMF. The flask was kept in thedark by covering it with an aluminum foil and 2 drops of Et₃N wereadded. Reaction was stirred at RT and was monitored by RP-HPLC. Uponcompletion, the solvent was evaporated under reduced pressure and theresulting yellow solid was dissolved in 1.5 ml of MeOH. Potassiumcarbonate (45 mg, 0.33 mmol) was added and removal of sugar acetates wasmonitored by RP-HPLC. Upon completion the product was purified byRP-HPLC (30-100% ACN in water, 20 min) to afford 83 mg (82% yield) ofyellow solid. The product was isolated as a mixture of diastereomers. ¹HNMR (400 MHz, DMSO): δ 8.88 (d, J=6.7 Hz, 4H), 8.77 (s, 1H), 8.63 (s,1H), 8.34 (s, 2H), 8.29-8.21 (m, 6H), 7.81 (d, J=7.9 Hz, 2H), 7.53 (d,J=16.2 Hz, 2H), 7.44 (s, 1H), 7.36-7.30 (m, 3H), 5.04-4.88 (m, 1H),4.70-4.50 (m, 3H of the sugar moiety, under broad H₂O peak), 4.28 (s,6H), 4.06 (s, 2H), 3.72 (d, J=2.7 Hz, 1H), 3.65-3.37 (m, 10H), 3.10-2.99(m, 3H), 2.83 (s, 1H), 2.24 (d, J=12.0 Hz, 1H), 1.96-1.21 (m, 12H). ¹³CNMR (100 MHz, DMSO): δ 166.40, 165.56, 157.25, 152.45, 145.25, 144.02,140.35, 135.01, 132.47, 132.03, 128.54, 128.46, 127.54, 126.62, 125.49,124.35, 124.03, 123.67, 117.95, 117.82, 111.55, 101.01, 100.31, 95.30,75.56, 73.58, 70.25, 67.98, 60.14, 49.22, 46.99, 40.53, 35.91, 33.21,32.90, 31.93, 31.78, 31.06, 30.75, 25.52, 25.17 (TFA signals are notshown). MS (ES+): m/z calc. for C₅₇H₆₂ClN₄O₁₂ ⁺ (quinone form): 1029.4;found: 1029.8.

In Vivo Evaluations.

All animal procedures were performed in compliance with Tel-AvivUniversity, Sackler School of Medicine guidelines and protocols approvedby the institutional animal care and use committee.

Six 7-weeks old BALB/c female mice (Harlan Laboratories Israel Ltd.,Jerusalem, Israel) were anesthetized using a mixture of ketamine (100mg/kg) and xylazine (12 mg/kg) injected subcutaneously. Then, mice wereinjected intraperitoneally or subcutaneously with 50 μL of the probesolution, previously incubated in PBS 7.4 (in the presence or absence ofβ-galactosidase) for 30 minutes. The mice were imaged andchemiluminescence was monitored for up to 15 min by intravitalnon-invasive bioluminescence imaging system (Photon Imager; BiospaceLab, Paris, France). Images were obtained by Photo-Acquisition software(Biospace Lab) and analyzed by M3Vision Software (Biospace Lab).

Optical imaging holds several advantages over other imaging modalities(e.g., radiography, magnetic resonance imaging and ultrasound).Fluorescent molecular probes at the NIR range, possess good spatialresolution and greater depth penetration than other wavelengths. In vivoimaging is required for determining the limit of detection and signalpenetration in live tissues. This data cannot be obtained by in vitromethods. These preliminary experiments use the minimal number of animalsto evaluate our new probe in terms of proof of concept (Redy-Keisar etal., 2015b). At the end of the experiment, mice were euthanized bycervical dislocation.

Chemiluminescence Microscopy Imaging of β-Galactosidase Activity.

Chemiluminescence images were acquired using Olympus LV200 invertedmicroscope fitted with an EMCCD camera (Hamamatsu C9100-13). HEK293 LacZstable cells (amsbio SC003) and HEK293-WT cells (control) were grown on35 mm glass bottom petri dishes at 37° C. for 24 h. Cell culture mediumwas changed to Molecular Probes® Live Cell Imaging Solution containing 5μM of Probe 3. Cells were incubated for another 20 minutes at 37° C.Thereafter, images were recorded with 20 minutes exposure time.

Results and Discussion

Design and Synthesis of Fluorophore-Tethered Dioxetane Probes.

The general structure of the dioxetane-fluorophore conjugate and itsactivation mechanism are presented in Scheme 8. Removal of the triggerby the analyte of interest initiates the CIEEL mechanism, which leads toenergy transfer from the excited benzoate to the dye, resulting inexcitation of the fluorophore. Thus, light emission should occur from ahighly emissive species (the fluorophore) at its correspondingwavelength.

We sought to develop a practical synthetic pathway that allows modularattachment of a fluorophore to the phenolic ring. Dioxetane is usuallyprepared by reaction of singlet oxygen with a double bond. Sinceconditions for production of singlet oxygen are not always compatiblewith the presence of a fluorophore, we developed a late-stagefunctionalization chemistry that allows attachment of the fluorophoreafter preparation of the dioxetane. The synthesis of adioxetane-fluorophore conjugate designed for activation byβ-galactosidase (as a model enzyme) is shown in Scheme 9. Commerciallyavailable aldehyde 1a was protected with trimethyl orthoformate to giveacetal 1b, followed by additional protection of the phenol group withTBSCl to afford compound 1c. The latter was reacted withtrimethylphosphite to produce phosphonate 1d, which was condensed with2-adamantanone via the Wittig-Horner reaction to give enol ether 1e.Deprotection of the TBS group of 1e gave phenol 1f, which was alkylatedwith a bromo-galactose derivative to afford compound 1g. Hartwig-MiyauraC—H borylation (Ishiyama et al., 2002) of 1g afforded phenylboronicester 1h, which was coupled with benzyl bromide 1i via Suzuki couplingreaction to give compound 1j. Oxidation of 1j with singlet oxygen gaveNHS-ester-functionalized dioxetane 1k. This NHS-ester readily reactswith various amine-functionalized dyes to afford dioxetane-fluorophoreconjugate 1m.

Following the synthetic strategy presented in Scheme 9, we preparedthree different chemiluminescent probes (Probes 1-3, see Schemes 5-7)for monitoring of β-galactosidase activity. Probes 2 and 3 are composedof dioxetane tethered with the fluorogenic dyes fluorescein and QCy(Karton-Lifshin et al., 2011; Karton-Lifshin et al., 2012),respectively. Probe 1 is a basic Schaap-dioxetane without a tethereddye. The chlorine substituent on the phenolic ring was introduced inorder to decrease the pKa of the phenol released after cleavage of theβ-galactosidase substrate. Such a pKa should allow the chemiexcitationpathway of the dioxetane to occur under physiological conditions.

Light-Induced Decomposition of Dioxetane-Dye Conjugates.

While working on the synthesis of the dioxetane-fluorophore conjugates,we encountered an unexpected phenomenon. Although Probe 1 seemed to bephotostable, Probes 2 and 3 appeared to decompose under normal roomillumination conditions. We measured the photostability of the probes inaqueous solution (PBS, pH 7.4) under normal room illumination. Thelight-induced decomposition was monitored over several hours by aRP-HPLC assay (FIG. 2).

Probe 1 was unaffected by light over a 12 h period. Probe 3 exhibitedsignificantly higher photostability than Probe 2. The light-induceddecomposition half-life of Probe 2 was 45 min, whereas the half-life ofProbe 3 was about 6 h. No decomposition of any of the probes wasobserved when the solutions were kept in the dark. A possible mechanismfor lightinduced decomposition of the dioxetane-fluorophore conjugatesmight involve electron transfer from the excited dye to theperoxy-dioxetane bond (Wakimoto et al., 2015). FIG. 3 illustrates apossible light-induced decomposition mechanism. The fluorophore ofconjugate I is excited by visible light to form excited species II.Electron transfer from the LUMO of the excited fluorophore to theantibonding σ* orbital of O—O peroxide bond results in bond cleavage andsubsequent decomposition of the dioxetane into benzoate III andadamantanone. The observed light instability of Probes 2 and 3underlines the advantage of our late-stage functionalization strategyover previous reported synthetic methods for dioxetane-fluorophoreconjugates. The oxidation of the enol ether to the dioxetane is usuallyperformed by singlet oxygen generated from oxygen by a light source anda photosensitizer. Such conditions, if applied after the conjugation ofthe fluorophore, could lead to decomposition of the dioxetane. We wereable to avoid light-induced decomposition by using late-stagefunctionalization chemistry that allows attachment of the fluorophoreonly after preparation of the dioxetane.

Energy Transfer Observed for Dioxetane-Dye Conjugate Vs. A Mixture ofDioxetane and a Dye.

We first wanted to evaluate the efficiency of energy transfer in thedioxetane-fluorophore conjugates in comparison to a 1:1 mixture of Probe1 and a dye, upon activation with β-galactosidase. The obtainedchemiluminescence emission spectra are shown in FIG. 4. In the case offluorescein, two emission maxima were obtained for the dioxetane-dyemixture (FIG. 4, panel A) at wavelengths of 470 and 535 nm. Thesewavelengths correspond to the direct chemiluminescence of Probe 1 and tothe emission of fluorescein resulting from energy transfer,respectively. On the other hand, Probe 2 (dioxetane-fluoresceinconjugate), upon activation by β-galactosidase, decomposed to emitgreenish light with maximum emission wavelength of 535 nm exclusively(FIG. 4, panel B). The observed chemiluminescence spectrum of the probewas almost identical to its fluorescence spectrum (dotted line);indicating a complete energy transfer to the fluorescein acceptor. Inthe case of QCy, only blue emission with maximum wavelength of 470 nmwas obtained for the dioxetane-dye mixture (FIG. 4, panel C). Thisemission corresponds to the direct chemiluminescence of Probe 1. On theother hand, Probe 3 (QCy-tethered dioxetane) decomposed to emit NIRlight with maximum emission wavelength of 714 nm (FIG. 4, panel D).Similarly, as observed for Probe 2, the chemiluminescence spectrum ofProbe 3 was found to be almost identical to its fluorescence spectrum(dotted line). This observation clearly supports the energy transfermechanism illustrated in Scheme 8, and properly demonstrates thesignificance of covalent conjugation between the dioxetane and the dye.

Chemiluminescence Parameters Measured for Probes 1, 2 and 3 and theirAbility to Detect and Image β-Galactosidase.

Next, we measured the light emission of the probes, as a function oftime, in the presence and in the absence of β-galactosidase. The probesexhibited a typical chemiluminescent kinetic profile in the presenceβ-galactosidase with an initial signal increase to a maximum followed bya slow decrease to zero (FIG. 5, panels A-C). No light emission wasobserved from the probes in the absence of β-galactosidase. FIG. 5,panels D-F, show the total photon counts emitted from each of theprobes. The measured chemiluminescence parameters obtained for Probes 1,2 and 3 are summarized in Table 8.

The chemiluminescence quantum yield (ϕ_(CL)) of probes 2 and 3 wascalculated using that of Probe 1 as a known standard (Edwards et al.,1994). Probes 2 and 3 exhibited significantly higher light emission thanthe emission exhibited by Probe 1 (114-fold for Probe 2 and 27-fold forProbe 3). In addition, Probes 2 and 3 had longer half-lives of lightemission under identical conditions.

TABLE 8 Chemiluminescent parameters obtained for Probes 1-3 Probeλ_(max) [nm] T_(1/2) [min] Φ_(CL) Relative Φ_(CL) 1 470  40 3.3 × 10⁻⁵ 12 535 170 3.8 × 10⁻³ 114 3 714 100 9.0 × 10⁻⁴ 27.2

Probe 2 exhibited brighter chemiluminescence than the other probes sinceits energy transfer is resulted with an excited fluorescein species (adye with 90% fluorescence quantum yield). We therefore selected Probe 2and demonstrated its ability to detect β-galactosidase (FIG. 6). Theprobe was incubated with different concentrations of β-galactosidase andtotal chemiluminescence emission was collected over 1 h period. Linearcorrelation was observed between enzyme concentrations and integratedchemiluminescence signal, enabling quantification of enzymeconcentration. We determined a detection limit (blank control+3 SD) of4.0×10⁻³ units/mL.

The ability of Probes 2 and 3 to image β-galactosidase activity wasinitially evaluated in aqueous solution (PBS, pH 7.4) using Probe 1 as acontrol (FIG. 7A). In the absence of β-galactosidase, nochemiluminescence was observed from any of the probes; however, in thepresence of the enzyme, Probes 2 and 3 emitted light with much strongerintensity than that of Probe 1 (about 100-fold for Probe 2 and 25-foldfor Probe 3, FIG. 7B). Probes 2 and 3 were selected for furtherevaluation in vivo; notably, Probe 3 emits light within the NIR region.This region of light is optimal for in vivo imaging applications sinceNIR photons penetrate organic tissues (Weissleder, 2001; Gnaim andShabat 2014; Kisin-Finfer et al., 2014; Redy-Keisar et al., 2014;Redy-Keisar et al., 2015a-b). Probes 2 and 3 were incubated withβ-galactosidase and then injected subcutaneously to mice. Under suchconditions, clear chemiluminescence images were obtained for both probes(FIG. 7C); however, the signal intensity obtained from Probe 3 was about6-fold higher than that obtained from Probe 2 (FIG. 7D). Nochemiluminescence signal was obtained from the probes withoutpreincubation with β-galactosidase.

In order to further compare the in vivo chemiluminescence signals ofProbes 2 and 3, the probes (with and without ex vivo incubation withβ-galactosidase) were injected into mice via the intraperitoneal route.Remarkably, Probe 3 produced a strong chemiluminescence in vivo image,whereas no chemiluminescence signal was observed for Probe 2 (FIG. 8).These observations clearly demonstrate the in vivo imaging advantage ofthe NIR chemiluminescence produced by Probe 3 vs. that of the greenwavelength produced by Probe 2.

In previous examples that used Schaap's dioxetanes for in vivo imaging(Cao et al., 2015; Cao et al., 2016; Liu and Mason, 2010), asurfactant-dye adduct (Emerald-II enhancer) was added to the injectedsolution in order to allow detection of the chemiluminescence signal.The use of multi-component system for in vivo imaging has its obviouslimitations, especially when the animal is treated systemically. As wedemonstrated in FIG. 8, bright image was obtained from the QCy-tethereddioxetane (Probe 3), following ex vivo activation with β-galactosidaseand intraperitoneal injection into mice. Here we established the proofof concept of the dioxetane-fluorophore conjugates to act as turn-ONchemiluminescent probes for in vivo imaging. In the next step, we intendto study the capability of the probes to image real pathological events,such as cancer and inflammation. Yet, to demonstrate imaging based onreal endogenous activity, we next sought to image cells thatendogenously overexpressed β-galactosidase.

Cell Imaging Using Chemiluminescence Microscopy by Probe 3.

While fluorescence microscopy is a well-known established method forcell imaging, bioluminescence microscopy has recently been emerged(Bauer, 2013). Instrumentation improvements led to the development ofthe LV200 microscope by Olympus. The setup of this microscope hassignificantly improved the ability to localize and quantify luminescenceprobes at single cell resolution. So far, only luciferin wasdemonstrated as a probe to image cells transfected by the luciferasegene. We sought to evaluate the ability of Probe 3 to image cells withoverexpression of β-galactosidase by using the LV200 microscope. HEK293(transfected by LacZ) and HEK293-WT (control) cells were incubated withProbe 3 and then imaged by the LV200 (FIG. 9) using a 20× objective (NA0.75). Probe 3 was able to produce chemiluminescence images of theHEK293-LacZ cells (FIG. 9, panel b), while no chemiluminescence signalat all was observed by the HEK293-WT cells (FIG. 9, panel d).

To improve the image quality, the HEK293-LacZ cells were fixed by using4% formaldehyde and permeabilized with 0.1% Triton X-100. The cells werethen incubated with Probe 3 and imaged by the microscope using a 60×objective (NA 1.42). As can be seen in FIG. 10 (panel a, transmittedlight; panel b, chemiluminescence), the cells became visible, exhibitinga clear chemiluminescence emission.

Although the quality of the obtained images is not high yet, as far aswe know, these are the first chemiluminescence cell images produced by aturn-ON small-molecule probe that is not related to luciferin. Thisapproach should allow imaging other enzymatic/chemical reactivities incells by replacing the triggering group of the probe with theappropriate analyte responsive substrates. The synthetic strategydeveloped in this work enables convenient synthesis of variouschemiluminescence probes. For example, a lipase or an esterase probe canbe prepared by incorporation of an appropriate ester triggering groupinstead of the β-galactose one. Similarly, the synthesis of proteasesprobes can be achieved by using a short specific peptide as triggeringsubstrate. Of course, incorporation of certain substrates could be morechallenging than others; however, the use of orthogonal protectinggroups should assist in solving synthetic difficulties.

Conclusions

In summary, we have developed a simple and practical synthetic route forpreparation of turn-ON fluorophore-tethered dioxetane chemiluminescentprobes. The effectiveness of the synthesis is based on a late-stagefunctionalization of a dioxetane precursor by Hartwig-Miyaura C—Hborylation, followed by subsequent Suzuki coupling and oxidation todioxetane. The obtained intermediate is composed of a reactiveNHS-ester-dioxetane ready for conjugation with any fluorophore-aminederivative. We also reported the phenomenon of light-induceddecomposition of dioxetane-fluorophore conjugates, which highlights theadvantage of our synthetic method. The chemiluminescent emission of thefluorophore tethered dioxetane probes was significantly amplified incomparison to a classic dioxetane probe through an energy transfermechanism. The synthesized probes produced light of various colors thatmatched the emission wavelength of the excited tethered fluorophore.Using our synthetic route, we synthesized two fluorophore-tethereddioxetane probes designed for activation by β-galactosidase andconjugated with green (fluorescein) and NIR (QCy) fluorescent dyes. Bothprobes were able to provide chemiluminescence in vivo images followingsubcutaneous injection after activation by β-galactosidase. However, achemiluminescence image following intraperitoneal injection was observedonly by the NIR probe. These are the first in vivo images produced bySchaap's dioxetane-based chemiluminescence probes with no need of anyadditive. The NIR probe was also able to image cells, bychemiluminescence microscopy, based on endogenous activity ofβ-galactosidase. Such probes could be used for in vivo imaging ofreporter genes, enzymes, and chemical analytes. We anticipate that ourpractical synthetic methodology for dioxetane-tethered building blockswill be useful for preparation of various chemiluminescent probessuitable for numerous applications.

Study 2. Chemiluminescence Cell Imaging by a Non-Luciferin BasedSmall-Molecule Probe: Striking Substituent Effect on the EmissiveSpecies

Experimental

General.

All reactions requiring anhydrous conditions were performed under anargon atmosphere. All reactions were carried out at RT unless statedotherwise. Chemicals and solvents were either A.R. grade or purified bystandard techniques. TLC: silica gel plates Merck 60 F254: compoundswere visualized by irradiation with UV light. Column chromatography:silica gel Merck 60 (particle size 0.040-0.063 mm), eluent given inparentheses. RP-HPLC: C18 5 u, 250×4.6 mm, eluent given in parentheses.Preparative RP-HPLC: C18 5 u, 250×21 mm, eluent given in parentheses.¹H-NMR spectra were recorded using Bruker Avance operated at 400 MHz.¹³C-NMR spectra were recorded using Bruker Avance operated at 100 MHz.Chemical shifts were reported in ppm on the δ scale relative to aresidual solvent (CDCl₃: δ=7.26 for ¹H-NMR and 77.16 for ¹³C-NMR,DMSO-d₆: δ=2.50 for ¹H-NMR and 39.52 for ¹³C-NMR). Mass spectra weremeasured on Waters Xevo TQD. Fluorescence and chemiluminescence wererecorded on Molecular Devices Spectramax i3x. Fluorescence quantum yieldwas determined using Hamamatsu Quantaurus-QY. All reagents, includingsalts and solvents, were purchased from Sigma-Aldrich.

Benzoate 5a.

2-Chloro-3-hydroxybenzaldehyde (1a, 312 mg, 2 mmol) was dissolved inMeOH (5 mL). Oxone (615 mg, 2 mmol) and In(OTf)₃ (112 mg, 0.22 mmol)were added at RT. The reaction mixture was heated to reflux andmonitored by RP-HPLC. After the reaction was completed the mixture wasfiltered and the filtrate was concentrated using a rotary evaporator.Purification by column chromatography (Hex:EtOAc 30:70) affordedbenzoate 5a as a white solid (339 mg, 92% yield). ¹H NMR (400 MHz,CDCl₃) δ 7.44 (dd, J=7.5, 1.8 Hz, 1H), 7.24 (t, J=7.5 Hz, 1H), 7.18 (dd,J=8.1, 1.8 Hz, 1H), 6.08 (s, 1H), 3.93 (s, 3H). ¹³C NMR (101 MHz, CDCl₃)δ 166.11, 152.59, 130.27, 127.87, 123.60, 119.74, 52.82. MS (ES−): m/zcalc. for C₈H₇ClO₃:186.0; found: 185.0 [M−H]⁻.

Compound 4b.

To a stirred solution of benzoate 4a (1.52 g, 10 mmol) in EtOH (4 mL)was added I₂ (1.02 g, 4 mmol) in one portion. The reaction was heated toreflux before an aqueous solution (2 mL) of HIO₃ (352 mg, 2 mmol) wasadded. The mixture was refluxed for 1 hour before it was cooled to RT.The product was recovered by filtration and washed with water to givecompound 4b as a white solid. (2.11 g, 76% yield). ¹H NMR (400 MHz,CDCl₃) δ 7.75 (d, J=8.2 Hz, 1H), 7.47 (d, J=1.9 Hz, 1H), 7.24 (dd,J=8.2, 1.9 Hz, 1H), 3.88 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 167.34,156.30, 139.41, 131.76, 122.61, 115.64, 91.51, 52.74. MS (ES−): m/zcalc. for C₈H₇ClIO₃:277.9; found: 276.9 [M−H]⁻.

Compound 4c.

A mixture of compound 4b (1.39 g, 5 mmol) and TEMP (1.52 μl, 0.05 mmol)in toluene (100 ml) was heated to 100° C. Then, SO₂Cl₂ (404 pd, 5 mmol)dissolved in toluene (50 ml) was added dropwise. The mixture was stirredat 100° C. for 1 hour. Upon completion, the reaction was cooled to RTand the product was recovered by filtration and washed with toluene togive compound 4c as a white solid (1.12 g, 72% yield). ¹H NMR (400 MHz,CDCl₃) δ 7.69 (d, J=8.3 Hz, 1H), 7.18 (d, J=8.3 Hz, 1H), 6.48 (s, 1H),3.92 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 165.57, 152.17, 137.40, 130.52,124.47, 118.97, 88.07, 52.98. MS (ES−): m/z calc. for C₈H₆ClIO₃:311.9;found: 310.9 [M−]⁻.

General Procedure: Heck Reaction of Iodophenols with Methyl Acrylate(Benzoates 6a and 7a).

Iodophenol (1 eq), methyl acrylate (3 eq) and Et₃N (4.2 eq) weredissolved in anhydrous ACN. Then Pd(OAc)₂ (0.05 eq) and P(o-tol)₃ (0.01eq) were added. The flask was sealed and the solution was stirred at120° C. Reaction was monitored by TLC (Hex:EtOAc 80:20). Uponcompletion, reaction mixture was diluted with EtOAc and washed withsaturated NH₄Cl. The organic phase was dried over Na₂SO₄ andconcentrated under reduced pressure. The residue was purified by columnchromatography on silica gel (Hex:EtOAc 85:15) to afford thecorresponding phenol acrylate.

Benzoate 6a.

Compound 4b (200 mg, 0.72 mmol) was reacted according to generalprocedure. The product was obtained as a white solid (130 mg, 77%yield). ¹H NMR (400 MHz, CDCl₃) δ 7.90 (d, J=16.2 Hz, 1H), 7.46-7.34 (m,3H), 6.60 (d, J=16.2 Hz, 1H), 3.83 (s, 3H), 3.78 (s, 3H). ¹³C NMR (101MHz, CDCl₃) δ 165.66, 149.22, 130.74, 130.17, 129.11, 127.97, 125.27,123.11, 121.36, 119.82, 52.99. MS (ES−): m/z calc. for C₁₂H₁₂O₅: 236.1;found: 235.1 [M−H]⁻.

Benzoate 7a.

Compound 4c (200 mg, 0.64 mmol) was reacted according to generalprocedure. The product was obtained as a white solid (115 mg, 67%yield). ¹H NMR (400 MHz, CDCl₃) δ 7.91 (d, J=16.2 Hz, 1H), 7.38 (d,J=8.2 Hz, 1H), 7.33 (d, J=8.2 Hz, 1H), 6.57 (d, J=16.2 Hz, 1H), 3.87 (d,J=0.5 Hz, 3H), 3.71 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 167.51, 165.49,151.22, 142.50, 138.57, 130.25, 126.93, 126.60, 123.18, 122.04, 52.95,52.19. MS (ES−): m/z calc. for C₁₂H₁₁ClO₅: 270.0; found: 269.1 [M−H]⁻.

General Procedure: Heck Reaction of Iodophenols with Acrylonitrile(Benzoates 8a and 9a).

Iodophenol (1 eq), acrylonitrile (3 eq) and Et₃N (1.5 eq) were dissolvedin anhydrous ACN. Then Pd(OAc)₂ (0.05 eq) was added and the flask wassealed. The mixture was heated to 120° C. under microwave irradiation.Reaction was monitored by TLC (Hex:EtOAc 80:20). Upon completion,reaction mixture was diluted with EtOAc and washed with saturated NH₄Cl.The organic phase was dried over Na₂SO₄ and concentrated under reducedpressure. The residue was purified by column chromatography on silicagel (Hex:EtOAc 80:20) to afford the corresponding phenol acrylate.

Benzoate 8a.

Compound 4b (200 mg, 0.72 mmol) was reacted according to generalprocedure. The product was obtained as a white solid (118 mg, 81%yield). ¹H NMR (400 MHz, CDCl₃) δ 7.55 (d, J=16.7 Hz, 1H), 7.45 (dd,J=8.1, 1.5 Hz, 1H), 7.42 (d, 1.5 Hz 1H), 7.32 (d, J=8.1 Hz, 1H), 6.27(d, J=16.7 Hz, 1H), 3.87 (s, 3H). ¹³C NMR (101 MHz, MeOD) δ 165.04,155.39, 144.50, 131.48, 127.72, 123.59, 118.79, 117.04, 115.07, 96.86,50.13. MS (ES−): m/z calc. for C₁₁H₉O₅: 203.1; found: 202.1 [M−H]⁻.

Benzoate 9a.

Compound 4c (200 mg, 0.64 mmol) was reacted according to the generalprocedure. The product was obtained as a white solid (1.04 mg, 69%yield). ¹H NMR (400 MHz, MeOD) δ 7.69 (d, J=16.8 Hz, 1H), 7.49 (d, J=8.2Hz, 1H), 7.28 (d, J=8.2 Hz, 1H), 6.41 (d, J=16.8 Hz, 1H), 3.90 (s, 1H).¹³C NMR (101 MHz, MeOD) δ 166.15, 152.77, 145.25, 132.97, 126.46,125.62, 121.47, 120.67, 118.23, 99.40, 52.00. MS (ES−): m/z calc. forC₁₁H₈ClNO₃: 237.0; found: 236.0 [M−H]⁻.

Compound 4e.

3-Hydroxybenzaldehyde dimethyl acetal 4d (Gopinath et al., 2002) (2580mg, 15.36 mmol) and imidazole (1568 mg, 23.04 mmol) were dissolved in 15ml of DCM. TBSCl (2764 mg, 18.42 mmol) was added and the solution wasstirred for 30 minutes at RT and monitored by TLC. Upon completion, thewhite precipitate was filtered-off and the solvent was evaporated underreduced pressure. Purification by column chromatography (Hex:EtOAc 95:5)afforded compound 4e as a colorless oil (4070 mg, 94% yield). ¹H NMR(400 MHz, CDCl₃): δ 7.26 (t, J=7.8 Hz, 1H), 7.04 (d, J=7.6 Hz, 1H), 6.94(t, J=2.0 Hz, 1H), 6.80 (dd, J=8.1, 2.3 Hz, 1H), 5.34 (s, 1H), 3.32 (s,6H), 0.99 (s, 9H), 0.20 (s, 6H). ¹³C NMR (100 MHz, CDCl₃): δ 155.73,139.71, 129.30, 120.22, 119.85, 118.58, 102.94, 52.74, 25.81, 18.32,−4.30.

Compound 4f.

Acetal 4e (4070 mg, 14.43 mmol) and trimethyl phosphite (2.56 ml, 21.65mmol) were dissolved in 40 ml of DCM. Reaction mixture was cooled to 0°C. and titanium(IV) chloride (2.38 ml, 21.65 mmol) was added dropwise.Reaction was monitored by TLC. Upon completion, the solution was pouredinto a saturated aqueous solution of NaHCO₃ (130 ml) at 0° C. After 10minutes of stirring, 100 ml of DCM was added and the phases wereseparated. Organic phase was dried over Na₂SO₄ and concentrated underreduced pressure. Purification by column chromatography (Hex:EtOAc30:70) afforded compound 4f as a colorless oil (3745 mg, 72% yield). ¹HNMR (400 MHz, CDCl₃): δ 7.21 (t, J=7.8 Hz, 1H), 6.99 (d, J=7.5 Hz, 1H),6.92 (d, J=1.9 Hz, 1H), 6.80 (d, J=8.1 Hz, 1H), 4.47 (d, J=15.6 Hz, 1H),3.68 (d, J=10.6 Hz, 3H), 3.64 (d, J=10.5 Hz, 3H), 3.36 (s, 3H), 0.96 (s,9H), 0.18 (s, 6H). ¹³C NMR (100 MHz, CDCl₃): δ 155.91, 135.59, 129.57,121.17, 120.54, 119.67, 80.88, 79.20, 58.66, 53.76, 25.74, 18.29, −4.39.MS (ES+): m/z calc. for C₁₆H₂₉O₅PSi: 360.1; found: 361.1 [M+H]⁺.

Compound 4g.

Phosphonate 4f (3745 mg, 10.38 mmol) was dissolved in 25 ml of anhydrousTHF under argon atmosphere at −78° C. LDA (2.0 M in THF, 6 ml, 12 mmol)was added and the solution was stirred for 20 minutes. A solution of2-adamantanone (1863 mg, 12.46 mmol) in 20 ml of THF was added, andafter 15 minutes of stirring at −78° C. reaction was allowed to warm toRT. Reaction was monitored by TLC. Upon completion, reaction mixture wasdiluted with EtOAc (150 ml) and washed with brine (150 ml). Organicphase was dried over Na₂SO₄ and concentrated under reduced pressure.Purification by column chromatography (Hex:EtOAc 95:5) afforded compound4g as a colorless oil (3200 mg, 80% yield). ¹H NMR (400 MHz, CDCl₃): δ7.09 (t, J=8.0 Hz, 1H), 6.89-6.84 (m, 2H), 3.30 (s, 3H), 3.27 (s, 1H),2.05 (s, 1H), 1.97-1.65 (m, 12H), 1.04 (s, 9H), 0.23 (s, 6H). ¹³C NMR(100 MHz, CDCl₃): δ 151.98, 140.24, 136.20, 130.69, 126.69, 126.58,124.93, 120.30, 56.98, 39.28, 39.14, 38.74, 37.32, 32.96, 29.70, 28.58,28.43, 25.86, 18.54, −4.28. MS (ES+): m/z calc. for C₂₄H₃₅ClO₂Si: 418.2;found: 419.3 [M+H]⁺.

Compound 4h.

Compound 4g (3200 mg, 8.3 mmol) was dissolved in 30 ml of THF.Tetrabutylammonium fluoride (1.0 M in THF, 9.2 ml, 9.2 mmol) was addedand the solution was stirred at RT. Reaction was monitored by TLC. Uponcompletion, reaction mixture was diluted with EtOAc (150 ml) and washedwith 1M HCl (100 ml). Organic phase was dried over Na₂SO₄ andconcentrated under reduced pressure. Purification by columnchromatography (Hex:EtOAc 85:15) afforded compound 4h as white solid(2130 mg, 95% yield). ¹H NMR (400 MHz, CDCl₃) δ 7.21 (t, J=7.8 Hz, 1H),6.88 (d, J=7.5 Hz, 1H), 6.82 (s, 1H), 6.79-6.71 (m, 1H), 5.30 (s, 1H),3.31 (s, 3H), 3.23 (s, 1H), 2.65 (s, 1H), 2.04-1.69 (m, 12H). ¹³C NMR(100 MHz, CDCl₃) δ 155.8, 142.8, 136.7, 132.4, 129.1, 121.8, 115.9,114.6, 57.7, 39.1, 39.0, 37.1, 32.2, 30.3, 28.2 ppm. MS (ES−): m/z calc.for C₁₈H₂₂O₂: 270.2; found: 269.3 [M−H]⁻.

Compound 4i.

Compound 4h (2130 mg, 7.9 mmol) was dissolved in 150 ml of Toluene andcooled to 0° C. N-Iodosuccinimide (1777 mg, 7.9 mmol) was added inportions. Reaction was monitored by TLC. Upon completion, reaction wasquenched with saturated Na₂S₂O₃, diluted with EtOAc (250 ml) and washedwith brine (200 ml). The organic phase was dried over Na₂SO₄ andconcentrated under reduced pressure. The residue was purified by columnchromatography on silica gel (Hex:EtOAc 85:15) to afford compound 4i asa white solid (2439 mg, 78% yield). ¹H NMR (400 MHz, CDCl₃) δ 7.62 (dd,J=8.1, 0.4 Hz, 1H), 6.96 (d, J=1.4 Hz, 1H), 6.65 (ddd, J=8.1, 1.8, 0.5Hz, 1H), 5.42 (d, J=0.6 Hz, 1H), 3.30 (t, J=2.4 Hz, 3H), 3.22 (s, 1H),2.63 (s, 1H), 2.00-1.67 (m, 12H). ¹³C NMR (101 MHz, CDCl₃) δ 154.66,142.32, 138.02, 137.84, 133.01, 123.68, 115.86, 84.21, 77.42, 77.10,76.79, 58.01, 39.22, 39.08, 37.16, 32.33, 30.33, 28.29. MS (ES−): m/zcalc. for C₁₈H₂₀ClIO₂: 396.1; found: 395.1 [M−H]⁻.

Compound 4j.

Compound 1f (2420 mg, 7.9 mmol) was dissolved in 150 ml of toluene andcooled to 0° C. N-Iodosuccinimide (1777 mg, 7.9 mmol) was added inportions. Reaction was monitored by TLC. Upon completion, the solventwas evaporated under reduced pressure. Purification by columnchromatography (Hex:EtOAc 80:20) afforded compound 4j as a white solid(1531 mg, 45% yield). ¹H NMR (400 MHz, CDCl₃) δ 7.61 (d, J=8.1 Hz, 1H),6.62 (d, J=8.1 Hz, 1H), 6.15 (s, 1H), 3.30 (s, 3H), 3.25 (s, 1H), 2.09(s, 1H), 2.01-1.64 (m, 12H). ¹³C NMR (101 MHz, CDCl₃) δ 151.17, 139.21,136.77, 135.75, 132.68, 125.27, 120.05, 82.22, 57.30, 39.10, 38.67,37.09, 32.91, 29.72, 28.38. MS (ES−): m/z calc. for C₁₈H₂₀ClIO₂: 430.0;found: 429.3 [M−H]⁻.

General Procedure: Heck Reaction of Iodophenols with Methyl Acrylate(Compounds 6c and 7c).

Iodophenol (1 eq), methyl acrylate (3 eq), and Et₃N (1.5 eq) weredissolved in anhydrous ACN. Then Pd(OAc)₂ (0.05 eq) and P(o-tol)₃ (0.01eq) were added. The flask was sealed and the solution was stirred at120° C. Reaction was monitored by TLC (Hex:EtOAc 80:20). Uponcompletion, reaction mixture was diluted with EtOAc and washed withsaturated NH₄Cl. The organic phase was dried over Na₂SO₄ andconcentrated under reduced pressure. The residue was purified by columnchromatography on silica gel (Hex:EtOAc 85:15) to afford thecorresponding phenol acrylate.

Compound 6c.

Compound 4i (395 mg, 1 mmol) was reacted according to the generalmethod. The product was obtained as a pale yellow solid (248 mg, 70%yield). ¹H NMR (400 MHz, CDCl₃) δ 7.99 (d, J=16.1 Hz, 1H), 7.43 (d,J=8.4 Hz, 1H), 6.95-6.74 (m, 2H), 6.74-6.45 (m, 2H), 3.82 (s, 3H), 3.33(s, 3H), 3.23 (s, 1H), 2.70 (s, 1H), 2.06-1.72 (m, 12H). ¹³C NMR (101MHz, CDCl₃) δ 168.57, 155.44, 142.50, 140.28, 138.92, 134.09, 128.94,122.22, 121.01, 118.11, 116.70, 58.17, 51.87, 39.30, 39.15, 37.17,32.45, 30.54, 28.30. MS (ES−): m/z calc. for C₂₂H₂₆O₄: 354.2; found:353.2 [M−H]⁻.

Compound 7c.

Compound 4j (430 mg, 1 mmol) was reacted according to the generalmethod. The product was obtained as a pale yellow solid (271 mg, 70%yield). ¹H NMR (400 MHz, CDCl₃) δ 7.94 (d, J=16.2 Hz, 1H), 7.38 (d,J=8.0 Hz, 1H), 6.86 (d, J=8.0 Hz, 1H), 6.61 (d, J=16.2 Hz, 1H), 6.28 (s,1H), 3.84 (s, 3H), 3.35 (s, 3H), 3.27 (d, J=4.9 Hz, 1H), 2.12 (s, 1H),2.02-1.66 (m, 12H). ¹³C NMR (101 MHz, CDCl₃) δ 232.43, 206.72, 173.51,167.74, 150.65, 139.23, 136.60, 132.96, 126.80, 123.82, 123.70, 121.97,121.54, 119.77, 95.27, 57.41, 51.86, 39.19, 38.89, 37.09, 32.95, 32.03,29.78, 28.37, 24.44. MS (ES−): m/z calc. for C₂₂H₂₅ClO₄: 388.14; found:387.4 [M−H]⁻.

General Procedure: Heck Reaction of Iodophenols with Acrylonitrile(Compounds 8c and 9c).

Iodophenol (1 eq), acrylonitrile (3 eq) and Et₃N (1.5 eq) were dissolvedin anhydrous ACN. Then Pd(OAc)₂ (0.05 eq) was added and the flask wassealed. The mixture was heated to 120° C. under microwave irradiation.Reaction was monitored by TLC (Hex:EtOAc 80:20). Upon completion,reaction mixture was diluted with EtOAc and washed with saturated NH₄Cl.The organic phase was dried over Na₂SO₄ and concentrated under reducedpressure. The residue was purified by column chromatography on silicagel (Hex:EtOAc 80:20) to afford the corresponding phenol acrylate.

Compound 8c.

Compound 4i (200 mg, 0.5 mmol) was reacted according to the generalmethod. The product was obtained as a pale yellow solid (129 mg, 80%yield). ¹H NMR (400 MHz, CDCl3) δ 7.81 (s, 1H), 7.61 (d, J=16.7 Hz, 1H),7.32 (d, J=8.0 Hz, 1H), 7.01 (s, 1H), 6.89 (d, J=7.9 Hz, 1H), 6.22 (d,J=16.7 Hz, 1H), 3.39 (s, 3H), 3.25 (s, 1H), 2.73 (s, 1H), 1.86 (m, J,12H). ¹³C NMR (101 MHz, CDCl₃) δ 156.44, 147.09, 142.23, 139.48, 135.29,129.47, 122.16, 120.55, 119.58, 116.79, 96.71, 77.68, 77.36, 77.05,58.45, 39.43, 39.29, 37.24, 32.69, 30.84, 29.98, 28.39. MS (ES−): m/zcalc. for C₂₁H₂₃NO₂: 321.17; found: 320.2 [M−H]⁻.

Compound 9c.

Compound 4j (200 mg, 0.45 mmol) was reacted according to the generalmethod. The product was obtained as a pale yellow solid (77 mg, 48%yield). ¹H NMR (400 MHz, CDCl₃) δ 7.47 (d, J=16.8 Hz, 1H), 7.16 (d,J=8.0 Hz, 1H), 6.77 (d, J=8.0 Hz, 1H), 6.36 (s, 1H), 6.07 (d, J=16.8 Hz,1H), 3.20 (s, 3H), 3.15 (s, 1H), 1.99 (s, 1H), 1.90-1.50 (m, 12H). ¹³CNMR (101 MHz, CDCl₃) δ 150.65, 145.45, 139.19, 137.55, 133.46, 126.82,123.84, 121.80, 121.12, 118.66, 98.63, 77.48, 77.16, 76.84, 57.49,38.82, 37.03, 32.97, 29.79, 28.31. MS (ES−): m/z calc. for C₂₁H₂₂ClNO₂:355.13; found: 354.3 [M−H]⁻.

Phenol 5b.

Enol ether 1f (100 mg 0.3 mmol) and few milligrams of methylene bluewere dissolved in 20 ml of DCM. Oxygen was bubbled through the solutionwhile irradiating with yellow light. The reaction was monitored byRP-HPLC. After completion, the reaction mixture was concentrated byevaporation under reduced pressure. The crude product was purified bypreparative RP-HPLC (gradient of ACN in water). The product was obtainedas a white solid. ¹H NMR (400 MHz, DMSO) δ 10.34 (s, 1H), 7.38 (d, J=7.2Hz, 1H), 7.27 (t, J=7.9 Hz, 1H), 7.08 (dd, J=8.0, 1.3 Hz, 1H), 3.07 (s,3H), 2.85 (s, 1H), 2.27 (d, J=12.2 Hz, 1H), 1.93 (s, 1H), 1.72-1.05 (m,11H). ¹³C NMR (101 MHz, DMSO) δ 154.23, 132.16, 127.40, 123.00, 118.02,111.59, 95.19, 49.14, 35.97, 33.23, 32.95, 31.82, 31.75, 31.12, 30.80,25.55, 25.24. (101 mg, 92% yield). MS (ES+): m/z calc. for C₁₈H₂₁ClO₄:336.1; found: 337.3 [M+H]⁺.

General Procedure: Dioxetane Formation (Compounds 6b-9b).

Enol ether and few milligrams of methylene blue were dissolved in 20 mlof DCM. Oxygen was bubbled through the solution while irradiating withyellow light. The reaction was monitored by RP-HPLC. After completion,the reaction mixture was concentrated by evaporation under reducedpressure. The crude product was purified by preparative RP-HPLC(gradient of ACN in water).

Phenol 6b.

Compound 6c (90 mg, 0.25 mmol) was reacted according to the generalprocedure. The product was obtained as a white solid (70 mg, 71% yield).¹H NMR (400 MHz, DMSO) δ 10.54 (s, 1H), 7.84 (d, J=16.2 Hz, 1H), 7.71(d, J=8.1 Hz, 1H), 7.19 (s, 1H), 6.96 (s, 1H), 6.67 (d, J=16.2 Hz, 1H),3.70 (s, 3H), 3.10 (s, 3H), 2.88 (s, 1H), 1.82-1.38 (m, 10H), 1.25 (d,J=12.9 Hz, 1H), 0.99 (d, J=12.8 Hz, 1H). ¹³C NMR (101 MHz, DMSO) δ167.56, 157.19, 139.75, 137.98, 129.52, 118.76, 111.80, 95.15, 52.00,50.26, 36.24, 34.65, 33.29, 32.97, 32.31, 31.62, 25.94, 25.81. MS (ES−):m/z calc. for C₂₂H₂₆O₆: 386.2; found: 385.2 [M−H]⁻.

Phenol 7b.

Compound 7c (60 mg, 0.15 mmol) was reacted according to the generalprocedure. The product was obtained as a white solid (20 mg, 31% yield).¹H NMR (400 MHz, DMSO) δ 10.14 (s, 1H), 7.91 (d, J=16.2 Hz, 1H), 7.80(d, J=8.4 Hz, 1H), 7.47 (d, J=8.3 Hz, 1H), 6.71 (d, J=16.1 Hz, 1H), 3.72(s, 3H), 3.09 (s, 3H), 2.86 (s, 1H), 2.22 (d, J=12.2 Hz, 1H), 1.79-1.27(m, 12H). ¹³C NMR (101 MHz, DMSO) δ 167.20, 139.09, 134.17, 126.77,124.05, 120.45, 111.89, 95.94, 52.16, 49.86, 36.47, 33.57, 32.48, 32.23,31.67, 31.42, 26.09, 25.78. MS (ES−): m/z calc. for C₂₂H₂₅ClO₆: 420.1;found: 419.2 [M−H]⁻.

Phenol 8b.

Compound 8c (100 mg, 0.31 mmol) was reacted according to the generalprocedure. The product was obtained as a white solid (55 mg, 50% yield).¹H NMR (400 MHz, DMSO) δ 10.76 (s, 1H), 7.65 (d, J=16.7 Hz, 2H), 7.18(s, 1H), 7.02 (s, 1H), 6.49 (d, J=16.8 Hz, 1H), 3.11 (s, 3H), 2.89 (s,1H), 2.03 (s, J=27.8, 9.6 Hz, 1H), 1.85-1.40 (m, 10H), 1.26 (d, J=13.2Hz, 1H), 0.93 (d, 1H). ¹³C NMR (101 MHz, DMSO) δ 172.34, 156.84, 146.10,138.37, 129.68, 119.63, 111.58, 97.96, 95.01, 55.30, 50.14, 36.07,34.49, 33.12, 32.82, 32.15, 31.45, 25.78, 25.64. MS (ES−): m/z calc. forC₂₁H₂₃NO₄: 353.2; found: 352.2 [M−H]⁻.

Phenol 9b.

Compound 9c (120 mg, 0.35 mmol) was reacted according to the generalprocedure. The product was obtained as a white solid (52 mg, 38% yield).¹H NMR (400 MHz, CDCl₃) δ 7.70 (d, J=8.1 Hz, 1H), 7.60 (d, J=16.8 Hz,1H), 7.42 (d, J=8.3 Hz, 1H), 6.61 (d, J=5.4 Hz, 1H), 6.23 (d, J=16.8 Hz,1H), 3.21 (s, 3H), 3.01 (s, 1H), 2.01 (s, 1H), 1.89-1.37 (m, 12H). ¹³CNMR (101 MHz, CDCl₃) δ 150.69, 144.77, 134.74, 126.82, 124.84, 122.80,118.18, 111.40, 99.99, 96.28, 49.68, 36.42, 34.01, 33.42, 32.79, 32.08,31.49, 26.04, 25.70. MS (ES−): m/z calc. for C₂₁H₂₁ClNO₄: 387.1; found:386.2 [M−H]⁻.

Compound 4l.

Compound 4k (Redy-Keisar et al., 2014) (1.0 g, 2.2 mmol) and NaI (1.0 g,6.7 mmol) were dissolved in 2 mL ACN and cooled to 0° C. After 10 min,TMS-Cl (837 μl, 6.7 mmol) was added. The reaction mixture stirred for 30minutes at RT and monitored by TLC (Hex:EtOAc 70:30). After completion,the reaction mixture diluted with EtOAc, and was washed with saturatedNH₄Cl. The organic layer was separated, washed with brine, dried overNa₂SO₄ and evaporated under reduced pressure. The crude product waspurified by column chromatography on silica gel (Hex:EtOAc 70:30) toafford compound 4l as a white solid (1.08 g, 87% yield). ¹H NMR (400MHz, CDCl₃) δ 7.31 (d, J=8.6 Hz, 2H), 6.91 (d, J=8.6 Hz, 2H), 5.56-5.35(m, 2H), 5.09 (dd, J=10.4, 3.4 Hz, 1H), 5.03 (d, J=7.9 Hz, 1H), 4.44 (s,2H), 4.25-4.02 (m, 1H), 2.18 (s, 3H), 2.04 (s, 6H), 2.02 (s, 3H). ¹³CNMR (101 MHz, CDCl₃) δ 170.45, 169.70, 156.61, 134.52, 130.36, 117.45,99.68, 71.33, 71.07, 68.82, 67.10, 61.64, 20.97, 5.67. MS (ES−): m/zcalc. for C₂₁H₂₅IO₁₀: 564.1; found: 587.2 [M+Na]⁺.

General Procedure for S_(N)2, Benzyl Ether Formation (Compounds 6d-9d).

Enol ether (1 eq) was dissolved in 1 mL dry DMF and cooled to 0° C.K₂CO₃ (1.2 eq) was added and the solution stirred at 0° C. for 10minutes, before compound 4l (1 eq) was added. The reaction mixturestirred for 30 minutes at RT and monitored by TLC (Hex:EtOAc 50:50).After completion, the reaction mixture diluted with EtOAc (100 ml) andwas washed with saturated NH₄Cl (100 ml). The organic layer wasseparated, washed with brine, dried over Na₂SO₄ and evaporated underreduced pressure. The crude product was purified by columnchromatography on silica gel (Hex:EtOAc 50:50).

Compound 6d.

Compound 6c (100 mg, 0.28 mmol) was reacted according to the generalprocedure. The product was obtained as a white solid (186 mg, 84%yield). ¹H NMR (400 MHz, CDCl₃) δ 8.03 (d, J=16.2 Hz, 1H), 7.49 (d,J=8.0 Hz, 1H), 7.36 (d, J=8.5 Hz, 2H), 7.02 (d, J=8.5 Hz, 2H), 6.94-6.88(m, J=7.5 Hz, 2H), 6.52 (d, J=16.1 Hz, 1H), 5.53-5.43 (m, 2H), 5.12 (d,J=3.4 Hz, 1H), 5.10 (s, 2H), 5.05 (d, J=7.9 Hz, 1H), 4.26-4.04 (m, 4H),3.78 (s, 3H), 3.25 (s, 3H), 3.23 (s, 1H), 2.63 (s, 1H), 2.18 (s, 3H),2.08 (d, J=5.6 Hz, 3H), 2.06 (s, 3H), 2.01 (s, 3H), 1.99-1.69 (m, 12H).¹³C NMR (101 MHz, CDCl₃) δ 170.48, 170.36, 170.25, 169.52, 168.06,157.08, 156.79, 142.95, 139.90, 139.03, 133.59, 131.46, 129.00, 128.38,122.85, 122.32, 118.09, 117.13, 113.32, 99.72, 71.11, 70.91, 69.82,68.66, 66.92, 61.41, 58.03, 51.70, 39.28, 39.11, 37.15, 32.41, 30.46,28.27, 20.83, 20.76, 20.69 MS (ES+): m/z calc. for C₄₃H₅₀O₁₄: 790.3;found: 813.6 [M+Na]⁺.

Compound 7d.

Compound 7c (200 mg, 0.51 mmol) was reacted according to the generalprocedure. The product was obtained as a white solid (273 mg, 65%yield). ¹H NMR (400 MHz, CDCl₃) δ 7.87 (d, J=16.2 Hz, 1H), 7.43-7.36 (m,3H), 7.03 (d, J=8.0 Hz, 1H), 6.97 (d, J=8.6 Hz, 2H), 6.41 (d, J=16.2 Hz,1H), 5.42-5.40 (m, J=3.7 Hz, 2H), 5.03 (d, 1H), 4.93-4.85 (m, 2H),4.21-4.09 (m, 4H), 3.75 (s, 3H), 3.26 (s, 3H), 3.23 (s, 1H), 2.09-1.62(m, 24H). ¹³C NMR (101 MHz, CDCl₃) δ 170.42, 170.32, 170.15, 169.47,167.12, 157.24, 153.68, 139.43, 138.86, 138.19, 132.43, 131.01, 130.45,130.11, 129.81, 129.64, 127.87, 125.13, 119.89, 116.99, 99.62, 75.62,72.79, 71.06, 70.85, 68.67, 66.96, 61.44, 60.42, 57.26, 51.83, 39.20,38.64, 37.05, 32.94, 29.70, 28.35, 20.76, 20.70, 14.23. MS (ES+): m/zcalc. for C₄₃H₄₉ClO₁₄: 824.3; found: 847.7 [M+Na]⁺.

Compound 8d.

Compound 8c (129 mg, 0.4 mmol) was reacted according to the generalprocedure. The product was obtained as a white solid (263 mg, 87%yield). ¹H NMR (400 MHz, CDCl₃) δ 7.59 (d, J=16.8 Hz, 1H), 7.41 (d,J=7.5 Hz, 1H), 7.31 (d, J=8.0 Hz, 2H), 7.26-7.24 (m, 2H), 7.05 (d, J=8.4Hz, 2H), 6.05 (d, J=16.8 Hz, 1H), 5.11 (m, J=8.7 Hz, 2H), 4.85 (d, J=7.6Hz, 1H), 3.98 (s, 1H), 3.88-3.77 (m, 2H), 3.62 (dd, J=7.9, 4.9 Hz, 2H),3.15 (s, 2H), 2.96 (s, 1H), 2.01 (s, 1H), 1.83-1.12 (m, 12H). ¹³C NMR(101 MHz, CDCl₃) δ 170.65, 170.55, 170.41, 169.73, 157.34, 157.22,146.30, 142.93, 140.22, 134.44, 131.06, 130.35, 129.53, 128.81, 122.57,122.05, 119.49, 117.42, 113.33, 99.81, 96.85, 71.33, 71.08, 70.23,68.84, 67.13, 61.63, 58.31, 39.46, 39.29, 37.30, 32.65, 30.69, 29.95,28.42, 20.95, 20.88. MS (ES−): m/z calc. for C₄₂H₄₇NO₁₂: 757.31; found:802.6 [M+HCOO]⁻.

Compound 9d.

Compound 9c (77 mg, 0.22 mmol) was reacted according to the generalprocedure. The product was obtained as a pale yellow solid (160 mg, 90%yield). ¹H NMR (400 MHz, CDCl₃) δ 7.32 (d, J=16.8 Hz, 1H), 7.19 (d,J=8.5 Hz, 2H), 7.16 (d, J=8.1 Hz, 1H), 6.92 (d, J=8.0 Hz, 1H), 6.87 (d,J=8.6 Hz, 2H), 5.73 (d, J=16.8 Hz, 1H), 5.37-5.26 (m, 2H), 5.00-4.92 (m,2H), 4.81 (d, J=6.5 Hz, 1H), 4.09-3.89 (m, 4H), 3.15 (s, 6H), 3.10 (s,1H), 2.01 (s, 4H), 1.93-1.45 (m, 21H). ¹³C NMR (101 MHz, CDCl₃) δ170.43, 170.33, 170.17, 169.53, 157.42, 153.30, 144.91, 139.31, 139.20,132.99, 130.58, 130.44, 130.13, 130.01, 128.87, 128.01, 124.52, 118.11,117.23, 99.64, 98.22, 75.72, 71.09, 70.86, 68.65, 66.95, 61.42, 57.40,39.21, 39.04, 38.65, 37.02, 33.01, 29.75, 28.33, 28.18, 20.81, 20.73,20.66. MS (ES−): m/z calc. for C₄₂H₄₆ClNO₁₂: 791.27; found: 836.8[M+HCOO]⁻.

Probe 5.

Was synthesized from compound 1g in Scheme 9, by deoxygenation of thedouble bond and removal of the acetyl groups from the galactose moiety.

General Procedure for Acetate Deprotection and Dioxetane Formation(Probes 6-9).

The acetate protected sugar-enol-ether was dissolved in MeOH (3 ml).Potassium carbonate (4.2 eq) was added and the solution was stirred atRT. The Reaction was monitored by RP-HPLC. Upon completion, the reactionmixture was diluted with EtOAc (100 ml) and washed with brine (100 ml).The organic phase was dried over Na₂SO₄ and concentrated under reducedpressure. The crude product was further reacted without purification.The crude product and few milligrams of methylene blue were dissolved in20 ml of DCM. Oxygen was bubbled through the solution while irradiatingwith yellow light. The reaction was monitored by RP-HPLC. Aftercompletion, the reaction mixture was concentrated by evaporation underreduced pressure. The crude product was purified by preparative RP-HPLC(gradient of ACN in water).

Probe 6.

Compound 6d (133 mg, 0.22 mmol) was reacted according to generalprocedure. The product obtained as a white solid (64 mg, 63% yield). ¹HNMR (400 MHz, MeOD) δ 8.01 (d, J=16.2 Hz, 1H), 7.66 (d, J=7.9 Hz, 1H),7.33 (d, J=8.1 Hz, 2H), 7.08 (d, J=8.3 Hz, 2H), 6.58 (d, J=16.2 Hz, 1H),5.24 (s, 2H), 4.81 (d, J=7.9 Hz, 1H), 3.87 (d, J=3.3 Hz, 1H), 3.81-3.68(m, 4H), 3.67-3.59 (m, 1H), 3.54 (dd, J=9.7, 3.3 Hz, 1H), 3.12 (s, 3H),2.89 (s, 1H), 1.80-1.38 (m, 12H). ¹³C NMR (101 MHz, MeOD) δ 169.27,139.28, 138.05, 128.32, 119.56, 116.61, 111.59, 101.62, 95.28, 75.56,73.49, 70.85, 69.63, 68.78, 60.97, 48.89, 35.99, 34.35, 33.18, 32.62,31.91, 31.72, 31.25, 26.14, 25.87. MS (ES−): m/z calc. for C₃₄H₄₀O₁₂:654.3; found: 653.4 [M−H]⁻.

Probe 7.

Compound 7d (273 mg 0.33 mmol) was reacted according to generalprocedure. The product obtained as a white solid (90 mg, 40% yield). ¹HNMR (400 MHz, MeOD) δ 7.87 (d, J=8.4 Hz, 1H), 7.83 (d, J=16.3 Hz, 1H),7.74 (d, J=8.4 Hz, 1H), 7.33 (d, J=8.5 Hz, 2H), 7.09 (d, J=8.4 Hz, 2H),6.55 (d, J=16.2 Hz, 1H), 4.93 (s, 2H), 4.83 (d, J=8.4 Hz, 1H), 3.90 (d,J=3.3 Hz, 1H), 3.82-3.77 (m, 4H), 3.77-3.73 (m, 2H), 3.72-3.65 (m, 1H),3.57 (dd, J=9.7, 3.4 Hz, 1H), 3.17 (s, 3H), 2.95 (s, 1H), 1.97 (s, 1H),1.88-1.35 (m, 12H). ¹³C NMR (101 MHz, MeOD) δ 167.22, 158.35, 138.22,131.78, 130.48, 129.58, 128.64, 125.19, 120.57, 116.45, 101.57, 95.86,75.83, 75.64, 73.50, 70.89, 68.87, 61.07, 51.12, 48.65, 48.31, 36.26,33.71, 32.26, 31.82, 31.57, 31.31, 26.33, 25.96. MS (ES+): m/z calc. forC₃₅H₄₁ClO₁₂: 688.2; found: 711.5 [M+Na]⁺.

Probe 8.

Compound 8d (130 mg 0.17 mmol) was reacted according to generalprocedure. The product obtained as a white solid (69 mg, 65% yield). ¹HNMR (400 MHz, CDCl₃) δ 7.56 (d, J=16.8 Hz, 1H), 7.40 (d, J=7.9 Hz, 1H),7.28 (d, J=7.9 Hz, 2H), 7.21 (br, 1H), 7.07 (d, J=7.0 Hz, 2H), 6.01 (d,J=16.8 Hz, 1H), 5.02 (dd, J=22.2, 11.2 Hz, 2H), 4.90 (s, 1H), 4.21-3.66(m, 10H), 3.15 (s, 3H), 2.98 (s, 1H), 2.06 (s, 1H), 1.86-1.40 (m, 10H).¹³C NMR (101 MHz, CDCl₃) δ 157.56, 145.99, 139.54, 130.22, 129.73,128.92, 119.11, 117.39, 111.89, 101.55, 98.18, 95.91, 74.65, 73.60,71.25, 70.73, 61.59, 50.29, 36.52, 35.03, 33.47, 32.51, 31.95, 31.76,26.21, 26.06. MS (ES−): m/z calc. for C₃₄H₃₉NO₁₀: 621.3; found: 644.4[M+Na]⁺.

Probe 9.

Compound 9d (160 mg, 0.2 mmol) was reacted according to generalprocedure. The product obtained as a white solid (61 mg, 46% yield). ¹HNMR (400 MHz, MeOD) δ 7.86 (d, J=8.3 Hz, 1H), 7.66 (d, J=8.4 Hz, 1H),7.45 (d, J=16.9 Hz, 1H), 7.26 (d, J=8.4 Hz, 2H), 7.10 (d, J=8.4 Hz, 2H),6.17 (d, J=16.8 Hz, 1H), 4.98 (s, 2H), 3.90 (d, J=3.1 Hz, 1H), 3.84-3.63(m, 10H), 3.58 (dd, J=9.6, 3.1 Hz, 1H), 3.17 (s, 3H), 2.95 (s, 1H), 2.36(d, J 12.4 Hz, 1H), 1.94 (s, 1H), 1.87-1.45 (m, 10H). ¹³C NMR (101 MHz,MeOD) δ 156.95, 142.68, 134.37, 129.90, 129.08, 127.72, 127.11, 123.05,116.09, 115.07, 109.88, 100.02, 97.64, 94.30, 74.29, 74.10, 71.92,69.34, 67.33, 59.54, 47.13, 46.75, 46.54, 46.33, 46.11, 45.90, 45.69,45.48, 34.68, 32.16, 32.01, 30.76, 30.24, 30.02, 29.74, 24.76, 24.39. MS(ES+): m/z calc. for C₃₄H₃₈ClNO₁₀: 655.2; found: 700.5 [M+HCOO]⁻.

Compound 10a.

Enol ether 6c (500 mg, 1.41 mmol) and triethylamine (0.49 ml, 3.5 mmol)were dissolved in 5 ml of DCM and cooled to 0° C.Trifluoromethanesulfonic anhydride (0.29 ml, 1.7 mmol) was added.Reaction mixture was monitored by TLC. Upon completion, reaction mixturewas diluted with DCM (100 ml) and washed with brine (100 ml). Organicphase was dried over Na₂SO₄ and concentrated under reduced pressure.Purification by column chromatography (Hex:EtOAc 80:20) affordedcompound 7a as a yellow oil (562 mg, 82% yield). ¹H NMR (400 MHz, CDCl3)δ 7.86 (d, J=16.0 Hz, 1H), 7.67 (d, J=8.1 Hz, 1H), 7.37 (dd, J=8.1, 1.1Hz, 1H), 7.31 (d, J=1.4 Hz, 1H), 6.51 (d, J=16.0 Hz, 1H), 3.83 (s, 3H),3.32 (s, 3H), 3.25 (s, 1H), 2.69 (s, 1H), 2.02-1.74 (m, 12H). ¹³C NMR(101 MHz, CDCl₃) δ 166.44, 147.46, 141.38, 139.76, 135.98, 135.85,129.08, 127.75, 126.42, 122.66, 121.60, 58.27, 51.96, 39.06, 38.98,36.90, 32.30, 31.55, 30.54, 28.05, 22.61, 14.07. ¹⁹F NMR (376 MHz,CDCl₃) δ −73.69. MS (ES+): m/z calc. for C₂₃H₂₅F₃O₆S: 486.1; found:489.3 [M+H]⁺.

Compound 10b.

Compound 10a (562 mg, 1.16 mmol), bis(pinacolato)diboron (589 mg, 2.32mmol), potassium acetate (341 mg, 3.48 mmol),[1,1′-bis(diphenylphosphino) ferrocene] dichloropalladium(II) (170 mg,0.23 mmol) were dissolved in 20 ml of dry dioxane and stirred at 120° C.under argon. Reaction was monitored by RP-HPLC. Upon completion,reaction mixture was diluted with EtOAc (100 ml) and washed with Brine.Organic phase was dried over Na₂SO₄ and concentrated under reducedpressure. Purification by column chromatography (Hex:EtOAc 70:30)afforded compound 10b as a yellow oil (441 mg, 82% yield). ¹H NMR (400MHz, CDCl3) δ 8.53 (d, J=16.0 Hz, 1H), 7.77 (d, J=1.7 Hz, 1H), 7.66 (d,J=8.1 Hz, 1H), 7.37 (dd, J=8.1, 1.6 Hz, 1H), 6.39 (d, J=16.0 Hz, 1H),3.80 (s, 3H), 3.29 (s, 3H), 3.25 (s, 1H), 2.63 (s, 1H), 2.01-1.75 (m,12H), 1.38 (s, 12H). ¹³C NMR (101 MHz, CDCl3) δ 167.83, 145.63, 143.09,139.00, 136.84, 136.38, 133.18, 131.96, 125.41, 118.30, 84.28, 58.11,51.70, 39.21, 39.15, 37.27, 32.38, 31.69, 30.39, 29.80, 28.36, 24.93,22.76, 14.24. MS (ES+): m/z calc. for C₂₈H₃₅O₇P: 464.3; found: 465.4[M+H]⁺.

Probe 10.

Borane 10b (441 mg, 0.95 mmol), NaOH 114 mg, 2.8 mmol) were dissolved in5 ml of 4:1 solution THF:H₂O. Reaction mixture was stirred at 40° C.overnight and was monitored by RP-HPLC. Upon completion, the reactionmixture diluted with EtOAc (100 ml) and was washed with saturatedsolution of 0.5M HCl (100 ml). The organic layer was separated, washedwith brine, dried over Na₂SO₄ and evaporated under reduced pressure. Thecrude residue and few milligrams of methylene blue were dissolved in 20ml of DCM. Oxygen was bubbled through the solution while irradiatingwith yellow light. The reaction was monitored by RP-HPLC. Uponcompletion, the solvent was concentrated under reduced pressure and theproduct was purified by preparative RP-HPLC (gradient of ACN in water).The product was obtained as a white solid (201 mg, 47% yield). ¹H NMR(400 MHz, MeOD) δ 8.53 (d, J=16.0 Hz, 1H), 7.81 (d, J=8.1 Hz, 1H), 6.41(d, J=16.0 Hz, 1H), 3.11 (s, 3H), 2.92 (s, 1H), 2.00 (s, 1H), 1.87-1.48(m, 12H), 1.32 (s, 12H). ¹³C NMR (101 MHz, MeOD) δ 169.03, 144.93,141.56, 135.21, 125.64, 120.20, 111.64, 95.07, 84.36, 74.54, 48.93,36.04, 34.43, 33.27, 32.61, 31.94, 31.76, 31.27, 29.46, 26.18, 26.00,24.85, 23.98, 23.84, 23.72. MS (ES−): m/z calc. for C₂₇H₃₅BO₇: 482.30;found: 399.2 [M-pinacol]⁻.

Compound 11a.

Enol ether 6c (200 mg, 0.56 mmol) was dissolved in 2 ml of DCM and DMAP(136 mg, 1.12 mmol) was added and the solution was stirred at RT.Triallyl phosphite (0.25 ml, 1.23 mmol) was dissolved in 2 ml of DCM andcooled to 0° C. Iodine (284 mg, 1.12 mmol) was added and reactionstirred to homogeneity. The iodine solution was pipetted to the phenolsolution. The reaction was monitored by TLC. Upon completion, reactionmixture was diluted with DCM (100 ml) and washed with brine (100 ml).The organic phase was dried over Na₂SO₄ and concentrated under reducedpressure. Purification by column chromatography (Hex:EtOAc 70:30)afforded compound 11a as a yellow oil (162 mg, 56% yield). ¹H NMR (400MHz, CDCl₃) δ 7.96 (d, J=16.1 Hz, 1H), 7.57 (d, J=8.1 Hz, 1H), 7.41-7.34(m, 1H), 7.17 (d, J=8.1 Hz, 1H), 6.47 (d, J=16.1 Hz, 1H), 6.05-5.86 (m,2H), 5.38 (dd, J=17.1, 1.4 Hz, 2H), 5.33-5.22 (m, 2H), 4.77-4.63 (m,4H), 3.81 (s, 3H), 3.33 (s, 3H), 3.24 (s, 1H), 2.70 (s, 1H), 2.03-1.75(m, 12H). ¹³C NMR (101 MHz, CDCl₃) δ 167.25, 149.04, 142.23, 139.25,138.13, 134.45, 131.97, 127.48, 126.09, 124.95, 121.20, 119.41, 118.89,69.12, 58.16, 51.78, 39.18, 39.05, 37.09, 32.33, 30.48, 28.20. ³¹P NMR(162 MHz, CDCl₃) δ −5.79. MS (ES+): m/z calc. for C₂₈H₃₅O₇P: 514.2;found: 537.3 [M+Na]⁺.

Probe 11.

Phosphate 11a (166 mg, 0.3 mmol) was dissolved in 1 ml of ACN.Pyrrolidine (0.153 ml, 1.86 mmol), triphenyl phosphine (16 mg, 0.06mmol), tetrakis(triphenylphosphine) palladium(0) (17 mg, 0.015 mmol) wasadded and the solution was stirred at RT. After completion theprecipitant was filtered and washed 3 times with ACN to give a yellowishsolid. The crude solid and NaOH (30 mg, 0.76 mmol) were dissolved in 2ml of 4:1 THF:H₂O solution. Reaction mixture was stirred at 40° C.overnight and was monitored by RP-HPLC. Upon completion, the reactionmixture was neutralized with 1M HCl_((aq)) and the solvent wasevaporated under reduce pressure. The crude residue and few milligramsof methylene blue were dissolved in 20 ml of DCM. Oxygen was bubbledthrough the solution while irradiating with yellow light. The reactionwas monitored by RP-HPLC. Upon completion the solvent was concentratedunder reduced pressure and the product was purified by RP-HPLC (10-75%ACN, ammonium carbonate 5 mM Buffer, 20 min) to afford Probe 11 as awhite solid (41 mg, 35% yield). ³¹P NMR (162 MHz, D₂O) δ −2.11. MS(ES−): m/z calc. for C₂₁H₂₅O₉P: 452.1; found: 451.2 [M−H]⁻.

Compound 12a.

Diethyl azodicarboxylate (95 μl, 0.6 mmol) was added to a cooled mixtureof compound 4m (Redy-Keisar et al., 2014) (184 mg, 0.5 mmol), compound6c (177 mg, 0.5 mmol) and triphenylphosphine (157 mg, 0.6 mmol) in 3 mLof THF at 0° C. Reaction was monitored by TLC (Hex:EtOAc 80:20). Uponcompletion, reaction mixture was diluted with EtOAc and washed withsaturated NH₄Cl. The organic phase was dried over Na₂SO₄ andconcentrated under reduced pressure. The residue was purified by columnchromatography on silica gel (Hex:EtOAc 80:20) to afford compound 12a asa pale yellow solid (274 mg, 78% yield). ¹H NMR (400 MHz, CDCl₃) δ 8.46(d, J=2.2 Hz, 1H), 8.36 (dd, J=8.7, 2.2 Hz, 1H), 8.02 (d, J=16.2 Hz,1H), 7.71 (d, J=8.7 Hz, 1H), 7.52 (d, J=7.9 Hz, 1H), 7.45 (d, J=8.4 Hz,2H), 7.29-7.20 (m, 2H), 6.95 (m, 2H), 6.52 (d, J=16.1 Hz, 1H), 5.15 (s,2H), 3.80 (s, 3H), 3.45 (s, 3H), 3.29 (s, 3H), 3.25 (s, 1H), 2.66 (s,1H), 1.99-1.76 (m, 12H). ¹³C NMR (101 MHz, CDCl₃) δ 168.00, 157.02,149.96, 148.45, 142.96, 139.73, 139.39, 139.29, 137.42, 136.69, 133.88,133.68, 128.75, 128.47, 127.97, 125.78, 122.96, 122.74, 119.50, 118.36,113.22, 69.66, 58.14, 53.54, 51.78, 39.94, 39.37, 39.18, 37.20, 32.54,31.71, 30.56, 29.82, 28.34, 22.77, 14.32, 14.23. MS (ES+): m/z calc. forC₃₆H₃₇N₃O₁₀S: 703.22; found: 726.4 [M+Na]⁺.

Probe 12.

Compound 12a (274 mg, 0.39 mmol) and few milligrams of methylene bluewere dissolved in 20 ml of DCM. Oxygen was bubbled through the solutionwhile irradiating with yellow light. The reaction was monitored by TLC(Hex:EtOAc 80:20). Upon completion, the reaction mixture wasconcentrated by evaporation under reduced pressure. The crude productwas purified by column chromatography on silica gel (Hex:EtOAc 80:20) toafford Probe 12 (255 mg, 89% yield). ¹H NMR (400 MHz, CDCl₃) δ 8.46 (d,J=2.0 Hz, 1H), 8.38 (dd, J=8.6, 2.1 Hz, 1H), 8.03 (d, J=16.2 Hz, 1H),7.72 (d, J=8.7 Hz, 1H), 7.60 (d, J=7.9 Hz, 1H), 7.47 (d, J=8.2 Hz, 3H),7.28 (d, J=7.4 Hz, 3H), 6.56 (d, J=16.2 Hz, 1H), 3.81 (s, 3H), 3.45 (s,4H), 3.20 (s, 2H), 3.02 (s, 1H), 2.10 (s, 1H), 1.91-1.36 (m, 16H). ¹³CNMR (101 MHz, CDCl₃) δ 167.70, 156.96, 149.98, 148.45, 139.48, 139.18,138.50, 137.11, 136.60, 133.67, 128.73, 127.97, 125.83, 124.80, 119.70,119.53, 111.84, 95.78, 69.91, 51.89, 50.12, 39.88, 36.43, 34.90, 33.41,33.30, 32.43, 31.83, 31.70, 31.64, 29.81, 26.10, 26.00, 22.77, 14.23. MS(ES+): m/z calc. for C₃₆H₃₇N₃O₁₂S: 735.21; found: 758.5 [M+Na]⁺.

Chemiluminescence Microscopy Imaging of f-Galactosidase Activity

Chemiluminescence images were acquired using Olympus LV200 invertedmicroscope fitted with an EMCCD camera (Hamamatsu C9100-13). HEK293 LacZstable cells (amsbio SC003) and HEK293-WT cells (control) were grown on35 mm glass bottom petri dishes at 37° C. for 24 h. Cell culture mediumwas changed to Molecular Probes® Live Cell Imaging Solution containing 5μM of probe 4. Cells were incubated for another 20 minutes at 37° C.Thereafter, images were recorded with 40 seconds exposure time.

Results and Discussion

In this Study, chemiluminescent probes based on the Schapp'sadamantylidene-dioxetane probe (Scheme 1), in which the phenolate donoris substituted at the ortho position of phenolic ring with a π* acceptorgroup such as methyl-acrylate and acrylonitrile, i.e., an electronacceptor or electron-withdrawing group, and optionally furthersubstituted at the other ortho position of the phenolic ring withchlorine, were designed and synthesized. Based on the teaching ofKarton-Lifshin et al. (2012) it has been postulated that suchdonor-acceptor pair design should potentially increase the emissivenature of the benzoate species. To the best of our knowledge, theinfluence of electron acceptor substituents on the aromatic moiety ofdioxetane chemiluminescence probes was never studied before forphysiologically-relevant pHs (Hagiwara et al., 2013; Matsumoto et al.,1996; Matsumoto et al., 2001; Matsumoto et al., 2002; Matsumoto et al.,2005).

To evaluate the substituent effect, numerous phenol-benzoate derivativeswith acceptor substituents at ortho and para positions of the phenolwere synthesized, and their fluorescent emission in PBS 7.4 weremeasured. The most significant effect was obtained when an acceptor wasincorporated at the ortho position of the phenol. Following broad screenof electron-withdrawing groups, we chose to focus on the chlorine,methyl-acrylate and acrylonitrile substituents. It was previously shownthat in order to enable the chemiexcitation mechanism underphysiological conditions, a chlorine substituent is introduced at theortho position of phenolic ring. This electron-withdrawing substituentdecreases the pKa of the phenol released after cleavage of theprotecting group and thereby enriches the relative concentration of thephenolate species in physiological pH. The methyl-acrylate andacrylonitrile substituents induced the highest increase in fluorescenceemission of their corresponded phenol-benzoates. The absorbance andfluorescence spectra of selected phenol-benzoate derivatives are shownin FIG. 11, and their molecular structure and spectroscopic parametersare summarized Table 9.

The emissive species generated by the chemiexcitation of commerciallyavailable adamantylidene-dioxetane probes are eventually the excitedstate of benzoates 4a or 5a. These benzoates do not present anymeasurable fluorescence under physiological conditions. However,incorporation of methyl-acrylate or acrylonitrile substituents at theortho position of the phenol (benzoates 6a and 8a) has produced strongfluorogenic phenol-benzoate derivatives (quantum yield; 0.5% and 7%respectively) with maximum emission wavelength of 550 nm and 525 nm,respectively. Insertion of additional chlorine substituent at the otherortho position (benzoate 7a) resulted in significant increase of thefluorescence emission intensity in comparison to its parent benzoate 6a(about 10-fold) with no change of the emission wavelength. Theintensification of fluorescence emission is attributed to the increaseconcentration of the phenolate species under physiological conditions,resulted from the electron-withdrawing effect of the chlorinesubstituent. Similar increase effect in fluorescence emission (about4-fold) was observed for benzoate 9a in comparison to its parentbenzoate 8a.

These results suggest that incorporation of the methyl-acrylate andacrylonitrile substituents (with or without the chlorine) in thedioxetane chemiluminescent luminophores could strengthen the emissivenature of the released benzoate. Such a substituent effect would lead toa significant increase of the dioxetane's chemiluminescence quantumyield under physiological conditions.

TABLE 9 Spectroscopic fluorescence parameters measured for selectedphenol-benzoate derivatives in PBS 7.4 Relative λ max_(ex) λ max_(em)fluorescence Phenol-benzoate [nm] [nm] ε _(400 nm) emissionΦFluorescence

290 270 ND negligible ND 4a

290 470 ND Negligible ND 5a

400 550 1200 1 0.5% 6a

400 550 7400 10.6 5.2% 7a

400 525 2200 4.7 7.0% 8a

400 525 7700 18.0 29.8% 9a

To test this hypothesis, we synthesized five differentadamantylidene-dioxetane luminophores with unmasked phenol functionalgroup (Table 10). Upon deprotonation of the phenol, the luminophoresundergo chemiexcitation decomposition to release the different benzoates(shown in Table 9) in their exited state. Next, we measured thechemiluminescence emission spectra and total light emission of theluminophores, under physiological conditions. The molecular structure ofthe dioxetane-luminophores and their chemiluminescence parameters aresummarized in Table 10. Predictably, the chemiluminescence emissionspectra of the dioxetane-luminophores overlap with the fluorescenceemission spectra of their corresponded benzoates (see FIG. 11).

TABLE 10 Chemiluminescence parameters of adamantylidene-dioxetanes 5b-9bRelative CL Dioxetane-luminophore π max_(CL) [nm] T_(1/2) min emissionΦ_(CL(H2O)) %

470 17 1 3.2 × 10⁻³ 5b

540 23 724 2.3 6b

540 7 780 2.5 7b

525 22 2295 7.4 8b

525 10 3043 9.8 9b

The dioxetane-luminophores exhibited a chemiluminescent exponentialdecay kinetic profile of with varied T₁/2. Dioxetane 5b, was used as areference compound as its chemiluminescence quantum yield under aqueousconditions is known (3.2×10⁻³%) (Trofimov et al., 1996; Edwards et al.,1994). As mentioned above, the chemiluminescence emission of dioxetane5b in water is extremely weak; however, dioxetane-luminophores 6b, 7b,8b and 9b exhibited remarkably strong chemiluminescence emission signalupon their deprotonation in PBS 7.4. Luminophore 6b (withmethyl-acrylate substituent) showed emission signal, which is about700-fold stronger than that of dioxetane 5b with chemiluminescencequantum yield of 2.3%. Luminophore 7b (with the methyl-acrylate andadditional chlorine substituent) showed similar signal enhancement withfaster kinetic profile relative to luminophore 6b (T_(1/2) of 7 min vs.23 min). Luminophore 9b (with the acrylonitrile and additional chlorinesubstituent) showed the highest enhancement of chemiluminescenceemission; about 3000-fold in comparison to that of dioxetane 5b withchemiluminescence quantum yield of 9.8%. Similar faster kinetic profilewas observed when the chlorine substituent was present on theluminophore (T_(1/2) of 10 min for dioxetane 9b vs. 22 min for dioxetane8b).

Turn-ON chemiluminescence probes can be simply prepared by masking ofthe phenol functional group of the dioxetane-luminophores with an enzymeresponsive substrate. To evaluate this option, we synthesized fivedifferent adamantylidene-dioxetane probes, using dioxetane-luminophores5b-9b, where the phenol is masked with a triggering substrate suitablefor activation by β-galactosidase (Probes 5-9, see Schemes 21-22). Toavoid steric interference (as a result of the ortho-substituent) at theenzyme cleavage-site, a short self-immolative spacer was installed inProbes 6-9, between the phenolic oxygen and the galactose substrate(Amir et al., 2003; Gnaim and Shabat, 2014; Sagi et al., 2008). Next, wemeasured the light chemiluminescence emission of the probes, as afunction of time, in the presence and in the absence of β-galactosidase.The kinetic profile of the probes' chemiluminescence signal and theirrelative emission intensities are shown in FIG. 12.

The probes exhibited a typical chemiluminescent kinetic profile in thepresence of β-galactosidase with an initial signal increase to a maximumfollowed by a slow decrease to zero. While Probes 6-9 exhibitedremarkably strong chemiluminescence emission signal under aqueousconditions in the presence of β-galactosidase, Probe 5 producedextremely weak emission (FIG. 12, inset). Probes 6 showed emissionsignal, which is about 500-fold stronger than that of Probe 5. Probe 7(with the chlorine substituent) showed similar signal enhancement withfaster kinetic profile relative to Probe 6. Similar faster kineticprofile was observed for Probe 9 in comparison to that of Probe 8. Probe9 showed the highest enhancement of chemiluminescence emission; about1800-fold, in comparison to that of Probe 5.

The striking enhancement of chemiluminescence emission obtained by thenew dioxetane-luminophores has promoted us to compare the signalintensity of Probe 9 to that of commercial chemiluminescence assays.There are several commercially available chemiluminescence probes basedon the adamantylidene-dioxetane. However, since the chemiluminescenceemission of these probes is very weak under aqueous conditions, asurfactant-dye adduct (enhancer) is usually added in order to amplifythe signal of the assay. The surfactant reduces water-induced quenchingby providing a hydrophobic environment for the chemiluminescent reactionthat transfers the emitted light to excite the nearby fluorogenic dye.Consequently, the low-efficiency luminescence process is amplifiedsignificantly in aqueous medium (Schaap et al., 1989). Commerciallyavailable Emerald-II™ enhancer (10%) was added to Probe 5 in thepresence of β-galactosidase (in PBS 7.4) and its chemiluminescenceemission was compared to that of Probe 6. The obtained results arepresented in FIG. 13. Emerald-II™ enhancer amplifies thechemiluminescence emission of Probe 5 by 248-fold (FIG. 13A).Remarkably, the chemiluminescence emission signal obtained by Probe 9 ismore than 8-fold stronger than that of Probe 5 with the Emerald-II™enhancer (FIG. 13B) under physiological conditions. This unprecedentedresult suggests that a simple small molecule dioxetane compound likeProbe 9 can produce chemiluminescence emission, which is about one orderof magnitude stronger than the signal produced by a two component-system(Probe 5 and Emerald-II™ enhancer). Since our probes produce underaqueous conditions relatively highly emissive benzoate species, additionof the Emerald-II™ enhancer had only mild amplification effect on theirchemiluminescence emission.

The activation of our chemiluminescence probes is based on removal of aprotecting group from the phenolic moiety. Therefore, differentphenol-protecting groups could be incorporated as triggering substratesfor various analytes/enzymes (Redy-Keisar et al., 2014). To demonstratethis modular feature, we synthesized three additional probes fordetection of the analytes hydrogen peroxide (Karton-Lifshin et al.,2011) and glutathione (GSH) and the enzyme alkaline-phosphatase (Schaapet al., 1989). Probe 10 was equipped with boronic-ester as a substratefor hydrogen peroxide, Probe 11 with phosphate group as a substrate foralkaline-phosphatase and Probe 12 with dinitro-benzene-sulfonyl group asa substrate for GSH (FIG. 14). The probes were prepared with an acrylicacid or methyl acrylate substituent at the ortho position of thephenolic oxygen. The presence of an ionizable carboxylic acid group hasincreased significantly their aqueous solubility of Probes 10 and 11 andenabled to conduct evaluation test at a relative high concentration. Atconcentration of 1 mM (pH 10), Probes 10 and 11 have produced brightgreen luminescence upon reaction with their analyte/enzyme. As describedabove, the probes decompose upon their activation to release the excitedstate of the corresponded benzoate. The acrylate substituent efficientlyincreases the emissive nature of the released benzoate to produce stronglight emission, clearly visible to the naked eye. Probe 12 hasrelatively moderate aqueous solubility with an applicable concentrationrange between 1-10 μM.

To evaluate the sensitively and selectivity of Probes 10, 11 and 12 todetect their corresponded analyte/enzyme, we determined the probes'limit of detection (LOD). The probes exhibited very good selectivitytowards their analyte of choice under physiological conditions (FIG.15). Probe 10 could detect hydrogen peroxide with an LOD value of 30 nM,Probe 11 could detect alkaline-phosphatase with an LOD value of 3.9μU/ml and Probe 12 could detect GSH with an LOD value of 1.7 μM.

Since chemiluminescence is generated through a kinetic profile, itssignal intensity, in absolute values, is often weaker in comparison tofluorescence. Therefore, to localize and quantify chemi/bio-luminescenceprobes at single cell resolution, a suitable microscope (like LV200 byOlympus) is required. We sought to evaluate the ability of our probes toimage cells that overexpressed β-galactosidase, by the LV200 microscope.After initial screening for cell-permeability, Probe 7 was selected forthe imaging evaluation. HEK293 (transfected by LacZ) and HEK293-WT(control) cells were incubated with Probe 7 and then imaged by the LV200(FIG. 16). The obtained images clearly support the probe in-vitroactivation by endogenously expressed β-galactosidase. Probe 7 was ableto produce high quality chemiluminescence images of the HEK293-LacZcells already with 20 second exposure time (FIG. 16b ). Nochemiluminescence signal at all was observed by the HEK293-WT cells(FIG. 16d ).

Over the past 30 years or so, numerous examples of chemiluminescenceprobes based on Schaap's dioxetane were reported in the literature(Bronstein et al., 1989; Stevenson et al., 1999; Sabelle et al., 2002;Richard et al., 2007; Richard et al., 2009; Turan and Akkaya, 2014; Caoet al., 2015; Cao et al., 2016; Clough et al., 2016). All of theseprobes were designed to release upon their activation the originalbenzoate, which is a weak emissive species under aqueous conditions. Inthis study, we aimed to redesign Schaap's dioxetane in order to developchemiluminescence probes that will be highly emissive in biologicalenvironment. As explained above, the chemiluminescence efficiency ofSchaap's dioxetane is essentially depended on the emissive nature of theobtained excite state benzoate. Thus, we initially sought to look at thefluorescence emission of different benzoates under physiologicalconditions. We assumed that if a substituent can increase the benzoate'sfluorescent emission in water, it will similarly be able to intensifythe chemiluminescence emission of a corresponded dioxetane probe underphysiological conditions. The obtained effect of the acrylate and theacrylonitrile substituents at the ortho position of the phenolic oxygenwas indeed incredible. With enhancement of greater than three orders ofmagnitude, we were able to obtain the most powerful chemiluminescenceadamantylidene-dioxetane probe known today suitable for use underaqueous conditions. Interestingly, introduction of the acrylatesubstituent at the para position of the phenolic oxygen, results withonly moderate effect on the fluorescence of the corresponded benzoateand on the chemiluminescence of the adamantylidene-dioxetane.

In commercial chemiluminescence assays signal enhancement is achievedindirectly, by energy transfer to a fluorescent dye (confined inmicelles that are shaped by a surfactant). Due to high toxicityconsequence, such multi-component probe system is obviously not suitablefor cell imaging (Partearroyo et al., 1990). The probes shown in theStudy are composed of single component, small molecule with direct modeof chemiluminescence emission and reasonable aqueous solubility. Suchcharacteristics make theses probes ideal for cell imaging applications.Probe 7 was able to provide excellent chemiluminescence cell imagesbased on endogenous β-galactosidase activity. As far as we know, theseare the first cell images that are obtained by non-luciferin smallmolecule based probe with direct chemiluminescence mode of emission.

The pKa value of phenolic benzoates is another factor that significantlyaffects the chemiluminescence emission of our probes. We have observedthat in order to enable the chemiexcitation mechanism underphysiological conditions, the pKa of the obtained phenol (after removalof the triggering group) should be around 8.5 or lower. A lower pKavalue for the phenol may be especially important for a probe aimed forin vitro use, when the probe penetrates into the cell throughendocytosis mechanism (the pH in the endosome is known to be around 6.5or lower). Probe 7 was selected for cell imaging evaluation as it iscomposed of a phenol with a pKa value of approximately 7.0.

The modular option to install different triggering substrate on thedioxetane probe, enables one to use our molecules to preparechemiluminescence probes for various analytes or enzymes of interest. Wehave demonstrated this option by synthesis and evaluation of four newchemiluminescence probes for detection of β-galactosidase,alkaline-phosphatase, hydrogen peroxide and ubiquitous thiols. The highchemiluminescence efficiency observed by theses probes under aqueousconditions should make them ideal substrates for biochemical tests inthe field of immunoassays.

Conclusions

In summary, we have developed a new molecular methodology to obtainchemiluminescence probes with high efficiency yield under physiologicalconditions. The methodology is based on the fluorescence emission effectof a substituent on the benzoate species obtained during thechemiexcitation pathway of the Schapp's adamantylidene-dioxetane probe.A striking substituent effect on the chemiluminescence efficiency of theprobes was obtained when acrylate and acrylonitrile electron-withdrawinggroups were installed. The chemiluminescence quantum yield of the bestprobe was greater than three orders of magnitude in comparison tostandard commercial available adamantylidene-dioxetane probe. So farbio/chemi-luminescence cell imaging was limited to luciferin relatedprobes. One of our new probes was able to provide high qualitychemiluminescence cell images based on endogenous activity ofβ-galactosidase. To date, this is the first demonstration ofcell-imaging achieved by non-luciferin small molecule based probe withdirect chemiluminescence mode of emission. The notion presented in thisstudy may further lead to development of new efficient chemiluminescenceprobes for various applications in the field of sensing and imaging.

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What is claimed is:
 1. A compound of the formula I:

wherein R¹ is selected from the group consisting of a linear or branched(C₁-C₁₈)alkyl, and (C₃-C₇)cycloalkyl; R² and R³ each independently isselected from the group consisting of a branched (C₃-C₁₈)alkyl and(C₃-C₇)cycloalkyl, or R² and R³ together with the carbon atom to whichthey are attached form a fused, spiro or bridged cyclic or polycyclicring; R⁵ and R⁶ each independently is selected from the group consistingof H, (C₁-C₁₈)alkyl, (C₂-C₁₈)alkenyl, (C₂-C₁₈)alkynyl,(C₃-C₇)cycloalkyl, and aryl, or R⁵ and R⁶ together with the oxygen atomsto which they are attached form a heterocyclic ring; R⁷, R⁸ and R⁹ eachindependently is H, or an electron acceptor group selected from thegroup consisting of halogen, —NO₂, —CN, —COOR¹⁰, —C(═O)R¹⁰ and —SO₂R¹⁰;R¹⁰ each independently is H or —(C₁-C₁₈)alkyl; and Q is a protectinggroup.
 2. The compound of claim 1, wherein: (i) R¹ is a linear orbranched (C₁-C₈)alkyl; or (ii) R² and R³ together with the carbon atomto which they are attached form a fused, spiro or bridged polycyclicring; or (iii) R⁵ and R⁶ each independently is (C₁-C₈)alkyl, andtogether with the oxygen atoms to which they are attached form aheterocyclic ring; or (iv) at least one of R⁷, R⁸ and R⁹ is H, and theother of R⁷, R⁸ and R⁹ each independently is an electron acceptor groupselected from the group consisting of halogen, —NO₂ and —CN; or (v) saidprotecting group is selected from the group consisting of —CH₃,—CH₂OCH₃, —C(═O)C(CH₃)₃, —CH₂—CH═CH₂, TBDMS, TBDPS, benzyl, and2-nitro-4,5-dimethoxybenzyl.
 3. The compound of claim 2, wherein R² andR³ together with the carbon atom to which they are attached formadamantyl.
 4. The compound of claim 2, wherein R⁵ and R⁶ each isisopropyl and together with the oxygen atoms to which they are attachedform 4,4,5,5-tetramethyl-1,3,2-dioxaborolanyl.
 5. The compound of claim1, wherein: R¹ is a linear or branched (C₁-C₈)alkyl; R² and R³ togetherwith the carbon atom to which they are attached form a fused, spiro orbridged polycyclic ring; R⁵ and R⁶ each independently is (C₁-C₈)alkyl,and together with the oxygen atoms to which they are attached form aheterocyclic ring; and at least one of R⁷, R⁸ and R⁹ is H, and the otherof R⁷, R⁸ and R⁹ each independently is an electron acceptor groupselected from the group consisting of halogen, —NO₂ and —CN.
 6. Thecompound of claim 5, wherein R² and R³ together with the carbon atom towhich they are attached form adamantyl; or R⁵ and R⁶ each is isopropyland together with the oxygen atoms to which they are attached form4,4,5,5-tetramethyl-1,3,2-dioxaborolanyl.
 7. The compound of claim 6,wherein R¹ is methyl; R² and R³ together with the carbon atom to whichthey are attached form adamantly; R⁵ and R⁶ each is isopropyl andtogether with the oxygen atoms to which they are attached form4,4,5,5-tetramethyl-1,3,2-dioxaborolanyl; R⁷, R⁸ and R⁹ are H; and Q isTBDMS.
 8. A composition comprising a carrier, and a compound accordingto claim 1.